FEBS Lett, 1997 Mar 10, 404(2-3), 179 - 84 Molecular cloning and expression of subunit 9 of the 26S proteasome; Hoffman L et al.; Seven peptides from subunit 9 (S9) of the human 26S proteasome were sequenced and this information was used to clone a HeLa cDNA that encodes the 46 kDa subunit . Rabbit polyclonal antisera were made against a ubiquitin fusion protein containing 12 amino acids from S9 and against a full-length S9 expressed in E . coli . Western blot analysis showed that the S9-specific antibodies bound the 26S proteasome and its regulatory complex separated on non-denaturing gels . In SDS-PAGE samples of the two complexes, the S9-specific antibodies bound a single 46 kDa subunit . Thus, a cDNA encoding a novel 26S protease subunit has been isolated, sequenced, and expressed. FEBS Lett, 1997 Mar 10, 404(2-3), 140 - 2 COOH-terminal decamers in proteins are non-random; Berezovsky IN et al.; We have undertaken an exhaustive statistical analysis of the amino acid sequences at the carboxyl-terminal (C) ends of proteins . The composition of the C-terminal decapeptides differs from that expected for the given proteins from the overall amino acid composition . For E . coli, yeast, and H . sapiens it was shown that positively charged amino acid residues are over-represented while Gly residues are under-represented . The C-terminal bias, a novel feature of protein structure, should be taken into account when molecular evolution, spatial structure, translational termination and protein folding are concerned. FEBS Lett, 1997 Mar 10, 404(2-3), 125 - 8 Amounts of proteins altered by mutations in the dnaA gene of Escherichia coli; Ohba A et al.; We identified proteins whose amounts were altered in a temperature-sensitive dnaA46 mutant of Escherichia coli . Proteins whose amounts were increased in the mutant were serine hydroxymethyltransferase, beta-ketoacyl {acyl carrier protein} synthase II, long-chain fatty acid transport protein, and UDP-glucose 4-epimerase, while the decreased ones were flagellin and D-ribose-binding protein . Transformation of the mutant with a plasmid containing the wild type dnaA gene complemented the phenotype . As pulse-labeling experiments revealed that the rates of synthesis of the proteins were altered in the mutant, DnaA protein may be involved in expression of these proteins. J Immunol Methods, 1997 Mar 10, 202(1), 49 - 57 A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation; Szarka RJ et al.; This study examines the intranasal instillation of lipopolysaccharide (LPS) into BALB/c mice causing acute pulmonary damage, due to neutrophil infiltration and sepsis . A dose response with LPS showed that an intranasal instillation of 167 microg/ml (10 microg/mouse) caused acute lung injury within 2-4 h and reached maximal damage at 24-48 h . We found the method of LPS administration for induction of acute pulmonary damage to be crucial . After 24 h post-LPS injection, a comparison showed a substantial increase in pulmonary damage with intranasal instillation of LPS . As for intravenous injection, it showed a baseline effect . This study indicates that LPS administered intranasally causes acute pulmonary damage, whereas with intravenous and intraperitoneal endotoxin administration a tissue-specific or similar degree of pulmonary injury may not develop. J Cell Biol, 1997 Mar 10, 136(5), 1091 - 7 MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts; Takenaka K et al.; In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor . To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti-Xenopus MAP kinase antibody . Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase-depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro . It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase-specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction . We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase-depleted extracts . But, adding recombinant Xenopus MAP kinase to the MAP kinase-depleted extracts restored the spindle assembly checkpoint . These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts . In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts . Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro. J Cell Biol, 1997 Mar 10, 136(5), 1081 - 90 Mitochondrial association of a plus end-directed microtubule motor expressed during mitosis in Drosophila; Pereira AJ et al.; The kinesin superfamily is a large group of proteins (kinesin-like proteins {KLPs}) that share sequence similarity with the microtubule (MT) motor kinesin . Several members of this superfamily have been implicated in various stages of mitosis and meiosis . Here we report our studies on KLP67A of Drosophila . DNA sequence analysis of KLP67A predicts an MT motor protein with an amino-terminal motor domain . To prove this directly, KLP67A expressed in Escherichia coli was shown in an in vitro motility assay to move MTs in the plus direction . We also report expression analyses at both the mRNA and protein level, which implicate KLP67A in the localization of mitochondria in undifferentiated cell types . In situ hybridization studies of the KLP67A mRNA during embryogenesis and larval central nervous system development indicate a proliferation-specific expression pattern . Furthermore, when affinity-purified anti-KLP67A antisera are used to stain blastoderm embryos, mitochondria in the region of the spindle asters are labeled . These data suggest that KLP67A is a mitotic motor of Drosophila that may have the unique role of positioning mitochondria near the spindle. J Mol Biol, 1997 Mar 7, 266(4), 733 - 44 Charged residues of the rotor protein FliG essential for torque generation in the flagellar motor of Escherichia coli; Lloyd SA et al.; The FliG protein of Escherichia coli is essential for assembly and function of the flagellar motor . Certain mutations in FliG give a non-motile, or Mot-, phenotype, in which flagella are assembled but do not rotate . Mutations with this property are clustered in a C-terminal segment of FliG that is stable when expressed alone, and thus probably constitutes an independently folded domain . Previously, we suggested that this domain forms the rotor portion of the active site for torque generation in the motor . In this work, we have used a mutational approach to identify the amino acid residues in the C-terminal domain of FliG that are most important for motor function . Site-directed mutagenesis was used to replace each of the conserved residues in this domain with alanine, and the effects on motor function were measured . Because charged residues have often been suggested to have important roles in torque generation, conserved charged residues were changed individually and in all pairwise combinations . The results show that three charged residues of FliG, Arg279, Asp286 and Asp287, are directly involved in torque generation . Mutations in these residues cause motility defects that suggest that they function jointly, in an active site whose most important property is a specific arrangement of charges . Two other charged residues, Lys262 and Arg295, may also be involved in torque generation, but are less critical than Arg279, Asp286 or Asp287 . Unchanged residues of the FliG motility domain do not appear to have direct roles in torque generation, although some are needed for the stability of the protein or for normal clockwise/ counter-clockwise switching . The Mot- mutations of fliG isolated previously by random mutagenesis do not alter the putative active-site residues, but render the proteins abnormally susceptible to proteolysis, suggesting significantly altered conformations or reduced stabilities. J Mol Biol, 1997 Mar 7, 266(4), 703 - 10 Sequence-dependent modulation of nucleotide excision repair: the efficiency of the incision reaction is inversely correlated with the stability of the pre-incision UvrB-DNA complex; Delagoutte E et al.; The UvrABC excinuclease is involved in the nucleotide excision repair (NER) pathway . Sequence-dependent differences in repair efficiency have been reported for many different lesions, and it is often suggested that sites with poor repair contribute to the occurrence of mutation hot spots . However, guanine bases modified by N-2-acetylaminofluorence (AAF) within the NarI site (5'-G1G2CG3CC-3') are incised by the UvrABC excinuclease with different efficiencies in a pattern not correlated with the potency of mutation induction . To gain insight into the mechanism of sequence-dependent modulation of NER, we analyzed the formation, the structure and the stability of UvrB-DNA pre-incision complexes formed at all three positions of the AAF-modified NarI site . We show that the efficiency of release of UvrA2 from specific UvrA2B-DNA complexes is sequence-dependent and that the efficiency of incision is inversely related to the stability of the pre-incision complex . We propose that the pre-incision complex, {UvrB-DNA}, when formed upon dissociation of UvrA2, undergoes a conformational change (isomerization step) giving rise to an unstable but incision-competent complex that we call {UvrB-DNA}' . The {UvrB-DNA} complex is stable and unable to form an incision-competent complex with UvrC . As the release of UvrA2, this isomerization step is sequence-dependent . Both steps contribute to modulate NER efficiency. J Mol Biol, 1997 Mar 7, 266(4), 677 - 87 Mechanistic insights into p53-promoted RNA-RNA annealing; Nedbal W et al.; The tumour suppressor protein p53 promotes the annealing of complementary nucleic acids in vitro . We observed an up to 1600-fold increase of RNA-RNA annealing by recombinant p53 protein which was shown to bind to RNA in sequence-independent way . Nuclease mapping experiments suggest that p53 binds to intramolecular duplex portions and only marginally changes the overall secondary structure of RNA at conditions of increased annealing . Thus, the mechanism of p53-promoted RNA-RNA annealing does not seem to be dependent on an activity that melts or changes RNA structure . The activation enthalpy of RNA-RNA annealing is decreased in the presence of p53, i.e . the p53 protein could stabilize the transition state whereas the activation entropy is unfavourable . A comparison with thermodynamic data measured for other facilitators strongly suggests that the mechanism of p53-promoted RNA-RNA annealing is distinct from the mechanism by which other facilitators work . The annealing activity of p53 is almost abolished in the presence of magnesium indicating that it can be sharply regulated in vitro and, in principle, could also be regulated in vivo. J Theor Biol, 1997 Mar 7, 185(1), 97 - 118 A functional model for the ribosome; Wood PN; The "ice" structure for the E . coli ribosome clearly illustrates how the ribosome could work and it is used as the basis of a detailed account of ribosome function . There have been objections to this structure as it does not fit with current beliefs about the position of the T-site, for the arriving tRNA, or the Exit-site: these beliefs are wrong . The elongation factor EF-Tu is required at both the T-site and the E-site, and these two attachment areas were combined into the false site . The E-site has been placed at the L1-ridge because deacylated tRNAs attach to protein L1, but none of these experiments used a cognate codon to get a positive identification of this as the E-site . The reason L1 attaches to deacylated tRNAs is that it is at the E-site in polysomes, which contain closely stacked, or overlapping, ribosomes with the L1-ridge at the interface between ribosomes . The revised data allows the 1989 ribosome model to be reconciled with the most contradictory results and it is then used to develop a more detailed picture of the ribosome. Biochim Biophys Acta, 1997 Mar 7, 1338(1), 77 - 92 Mobility of norbornane-type substrates and water accessibility in cytochrome P-450cam; Schulze H et al.; The behaviour of norbornane-type substrates bound to oxidised cytochrome P-450cam (CYP 101) in 60% (w/w) glycerol-containing phosphate buffer was investigated using electronic absorption spectroscopy . The high-pressure dependence study revealed that the value of the spin-state reaction-volume change decreased from -70 to -22.8 cm3/mol with decreasing high-spin state content from 99 to 63% . Simultaneously, the values for the enthalpy and entropy determined from the low-temperature dependence of the spin-state transition decreased from 73.7 to 24.3 kJ/mol and from 310.4 to 88.9 J/mol K, respectively . Under our experimental conditions the pH-value of the buffer remained at low temperatures and high pressures in the range of pH 7-8, in which no pH-value-induced spin-state conversion occurred . Therefore, the secondary effect of the temperature and pressure-induced pH change can be disregarded as being responsible for the observed spin-state transition effects . Substrate dissociation constants were determined . From the temperature-jump experiments (297 K to 180 K) we found a higher mobility in the active site for the substrates in the sequence (1R)-camphor, (1S)-camphor, camphane, (1R)- and (1S)-camphorquinone, norcamphor, and norbornane . Our findings can be explained by the incomplete fit of the methyl groups of the norbornane-type substrate to the protein, in particular to the I-helix, predominantly determining the substrate mobility and water accessibility to the protein. J Biol Chem, 1997 Mar 7, 272(10), 6766 - 76 Cloning and functional expression of a mammalian gene for a peroxisomal sarcosine oxidase; Reuber BE et al.; Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain . An additional H2O2-producing sarcosine oxidase has now been purified from rabbit kidney . A corresponding cDNA was cloned from rabbit liver and the gene designated sox . This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme . Sequence analysis revealed an N-terminal ADP-betaalphabeta-binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1 . Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases . Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver . Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney . Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway. J Biol Chem, 1997 Mar 7, 272(10), 6733 - 40 Cloning and expression of the cDNA encoding the human homologue of the DNA repair enzyme, Escherichia coli endonuclease III; Hilbert TP et al.; We previously purified a bovine pyrimidine hydrate-thymine glycol DNA glycosylase/AP lyase . The amino acid sequence of tryptic bovine peptides was homologous to Escherichia coli endonuclease III, theoretical proteins of Saccharomyces cerevisiae and Caenorhabditis elegans, and the translated sequences of rat and human 3'-expressed sequence tags (3'-ESTs) (Hilbert, T . P., Boorstein, R . J., Kung, H . C., Bolton, P . H., Xing, D., Cunningham, R . P., Teebor, G . W . (1996) Biochemistry 35, 2505-2511) . Now the human 3'-EST was used to isolate the cDNA clone encoding the human enzyme, which, when expressed as a GST-fusion protein, demonstrated thymine glycol-DNA glycosylase activity and, after incubation with NaCNBH3, became irreversibly cross-linked to a thymine glycol-containing oligodeoxynucleotide, a reaction characteristic of DNA glycosylase/AP lyases . Amino acids within the active site, DNA binding domains, and {4Fe-4S} cluster of endonuclease III are conserved in the human enzyme . The gene for the human enzyme was localized to chromosome 16p13.2-.3 . Genomic sequences encoding putative endonuclease III homologues are present in bacteria, archeons, and eukaryotes . The ubiquitous distribution of endonuclease III-like proteins suggests that the 5,6-double bond of pyrimidines is subject to oxidation, reduction, and/or hydration in the DNA of organisms of all biologic domains and that the resulting modified pyrimidines are deleterious to the organism. J Biol Chem, 1997 Mar 7, 272(10), 6671 - 6 The N-terminal moiety of CDC25(Mm), a GDP/GTP exchange factor of Ras proteins, controls the activity of the catalytic domain . Modulation by calmodulin and calpain; Baouz S et al.; This work describes the in vitro properties of full-length CDC25(Mm) (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21 . CDC25(Mm), isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p21.GDP dissociation rate; however, its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25(Mm285)) and 5 times lower than the activity of the C-terminal half-molecule (631 residues) . This reveals a negative regulation of the catalytic domain by other domains of the molecule . Accordingly, the GEF activity of CDC25(Mm) was increased severalfold by the Ca2+-dependent protease calpain that cleaves around a PEST-like region (residues 798-853), producing C-terminal fragments of 43-56 kDa . In agreement with the presence of an IQ motif on CDC25(Mm) (residues 202-229), calmodulin interacted functionally with the exchange factor . Depending on the calmodulin concentration an inhibition up to 50% of the CDC25(Mm)-induced nucleotide exchange activity on H-ras p21 was observed, an effect requiring Ca2+ ions . Calmodulin also inhibited C-CDC25(Mm285) but with a approximately 100 times higher IC50 than in the case of CDC25(Mm) ( approximately 10 microM versus 0.1 microM, respectively) . Together, these results emphasize the role of the other domains of CDC25(Mm) in controlling the activity of the catalytic domain and support the involvement of calmodulin and calpain in the in vivo regulation of the CDC25(Mm) activity. J Biol Chem, 1997 Mar 7, 272(10), 6539 - 47 Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone; Mittl PR et al.; The cysteine protease CPP32 has been expressed in a soluble form in Escherichia coli and purified to >95% purity . The three-dimensional structure of human CPP32 in complex with the irreversible tetrapeptide inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone was determined by x-ray crystallography at a resolution of 2.3 A . The asymmetric unit contains a (p17/p12)2 tetramer, in agreement with the tetrameric structure of the protein in solution as determined by dynamic light scattering and size exclusion chromatography . The overall topology of CPP32 is very similar to that of interleukin-1beta-converting enzyme (ICE); however, differences exist at the N terminus of the p17 subunit, where the first helix found in ICE is missing in CPP32 . A deletion/insertion pattern is responsible for the striking differences observed in the loops around the active site . In addition, the P1 carbonyl of the ketone inhibitor is pointing into the oxyanion hole and forms a hydrogen bond with the peptidic nitrogen of Gly-122, resulting in a different state compared with the tetrahedral intermediate observed in the structure of ICE and CPP32 in complex with an aldehyde inhibitor . The topology of the interface formed by the two p17/p12 heterodimers of CPP32 is different from that of ICE . This results in different orientations of CPP32 heterodimers compared with ICE heterodimers, which could affect substrate recognition . This structural information will be invaluable for the design of small synthetic inhibitors of CPP32 as well as for the design of CPP32 mutants. J Biol Chem, 1997 Mar 7, 272(10), 6406 - 15 The majority of stem cell factor exists as monomer under physiological conditions . Implications for dimerization mediating biological activity; Hsu YR et al.; Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer . We have determined a dimer association constant (Ka) of 2-4 x 10(8) M-1, using sedimentation equilibrium and size exclusion chromatography . SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum . Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form . When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Ka determination . Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized . The dimer Ka values, biophysical properties, and biological activities of these variants were studied . Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF . The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction. J Biol Chem, 1997 Mar 7, 272(10), 6361 - 9 Assembly and full functionality of recombinantly expressed dihydrolipoyl acetyltransferase component of the human pyruvate dehydrogenase complex; Yang D et al.; The dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) consists of 60 COOH-terminal domains as an inner assemblage and sequentially via linker regions an exterior pyruvate dehydrogenase (E1) binding domain and two lipoyl domains . Mature human E2, expressed in a protease-deficient Escherichia coli strain at 27 degrees , was prepared in a highly purified form . Purified E2 had a high acetyltransferase activity, was well lipoylated based on its acetylation, and bound a large complement of bovine E1 . Electron micrographs demonstrated that the inner core was assembled in the expected pentagonal dodecahedron shape with E1 binding around the inner core periphery . With saturating E1 and excess dihydrolipoyl dehydrogenase (E3) but no E3-binding protein (E3BP), the recombinant E2 supported the overall PDC reaction at 4% of the rate of bovine E2.E3BP subcomplex . The lipoates of assembled human E2 or its free bilipoyl domain region were reduced by E3 at rates proportional to the lipoyl domain concentration, but those of the E2.E3BP were rapidly used in a concentration-independent manner consistent with bound E3 rapidly using a set of lipoyl domains localized nearby . Given this restriction and the need for E3BP for high PDC activity, directed channeling of reducing equivalents to bound E3 must be very efficient in the complex . The recombinant E2 oligomer increased E1 kinase activity by up to 4-fold and, in a Ca2+-dependent process, increased phospho-E1 phosphatase activity more than 15-fold . Thus the E2 assemblage fully provides the molecular intervention whereby a single E2-bound kinase or phosphatase molecule rapidly phosphorylate or dephosphorylate, respectively, many E2-bound E1 . Thus, we prepared properly assembled, fully functional human E2 that mediated enhanced regulatory enzyme activities but, lacking E3BP, supported low PDC activity. J Biol Chem, 1997 Mar 7, 272(10), 6318 - 23 Excretion and uptake of putrescine by the PotE protein in Escherichia coli; Kashiwagi K et al.; The structure and function of the polyamine transport protein PotE was studied . Uptake of putrescine by PotE was dependent on the membrane potential . In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential (Kashiwagi, K., Miyamoto, S., Suzuki, F., Kobayashi, H., and Igarashi, K . (1992) Proc . Natl . Acad . Sci . U . S . A . 89, 4529-4533) . The Km values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 microM, respectively . Uptake of putrescine was inhibited by high concentrations of ornithine . This effect of ornithine appears to be due to putrescine-ornithine antiporter activity because it occurs only after accumulation of putrescine within cells and because ornithine causes excretion of putrescine . Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein . Both the NH2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and beta-galactosidase by various PotE-fusion proteins . The activities of putrescine uptake and excretion were studied using mutated PotE proteins . It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities . These three glutamic acids are located on the cytoplasmic side of PotE, and the function of these three residues could not be replaced by other amino acids . Putrescine transport activities did not change significantly with mutations at the other 13 glutamic acid or aspartic acid residues in PotE. J Biol Chem, 1997 Mar 7, 272(10), 6285 - 90 Active site topologies and cofactor-mediated conformational changes of nitric-oxide synthases; Gerber NC et al.; The active site topologies of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric-oxide synthases heterologously expressed in Escherichia coli have been examined using three aryldiazene (Ar-N=NH) probes . The topological information derives from (a) the rate and extent of aryl-iron complex formation in the presence and absence of tetrahydrobiopterin (H4B), Ca2+-dependent calmodulin (CaM), and L-arginine, and (b) the N-phenylprotoporphyrin IX regioisomer ratios obtained upon migration of the phenyl of the phenyl-iron complex to the heme nitrogen atoms . The N-phenylprotoporphyrin ratios indicate that the three NOS isoforms have related active site topologies with unencumbered space above all four pyrrole rings but particularly above pyrrole ring D . H4B binds directly above the heme pyrrole ring D or causes a conformational change that constricts that region, because H4B markedly decreases phenyl migration to pyrrole ring D . Small CaM-dependent changes in the nNOS N-phenylporphyrin isomer pattern are consistent with a conformational link between the CaM and heme sites in this protein . The ceiling height directly above the heme iron atom differs among the isoforms and is lower than in the P450 enzymes because only nNOS and iNOS react with 2-naphthyldiazene, and none of the isoforms reacts with p-biphenyldiazene . L-Arg blocks access to the heme iron atom in all three NOS isoforms and nearly suppresses the phenyldiazene reaction . The data indicate that topological differences, including differences in the size of the active site, are superimposed on the structural similarities among the NOS active sites. J Biol Chem, 1997 Mar 7, 272(10), 6220 - 5 Conformational and functional differences between recombinant human lens alphaA- and alphaB-crystallin; Sun TX et al.; Human and other mammalian lens proteins are composed of three major crystallins: alpha-, beta-, and gamma-crystallin . alpha-Crystallin plays a prominent role in the supramolecular assembly required to maintain lens transparency . With age, the crystallins, especially alpha-crystallin, undergo posttranslational modifications that may disrupt the supramolecular assembly, and the lens becomes susceptible to other stresses resulting in cataract formation . Because these modifications occur even at a relatively young age, it is difficult to obtain pure, unmodified crystallins for in vitro experiments . alpha-Crystallin is composed of two subunits, alphaA and alphaB . Before the application of recombinant DNA technology, these two alpha-crystallin subunits were separated from calf lens in the denatured state and reconstituted by the removal of the denaturant, but they were not refolded properly . In the present studies, we applied the recombinant DNA technology to prepare native, unmodified alphaA- and alphaB-crystallins for conformational and functional studies . The expressed proteins from Escherichia coli are in the native state and can be studied directly . First, alphaA and alphaB cDNAs were isolated from a human lens epithelial cell cDNA library . The cDNAs were cloned into a pAED4 expression vector and then expressed in E . coli strain BL21(DE3) . Pure recombinant alphaA- and alphaB-crystallins were obtained after purification by gel filtration and DEAE liquid chromatography . They were subjected to conformational studies involving various spectroscopic measurements and an assessment of chaperone-like activity . alphaA- and alphaB-crystallins have not only different secondary structure, but also tertiary structure . 1-Anilino-8-naphthalene sulfonate fluorescence indicates that alphaB-crystallin is more hydrophobic than alphaA-crystallin . The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for alphaB- than for alphaA-crystallin . The resulting data provide a base line for further studies of human lens alpha-crystallin. Arch Microbiol, 1997 Mar 7, 167(2/3), 143 - 50 The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase Miksch G, Fiedler E, Dobrowolski P, Friehs K. Heterologous gene products produced by Escherichia coli cells can be exported into the culture medium by the action of the kil gene of the ColE1 plasmid, which encodes a bacterial release protein . The kil gene was fused with the stationary-phase promoter of the fic gene of E . coli, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed . Using the gene for beta-glucanase (bgl) as a target gene, it was shown that the protein produced was only secreted into the medium during the stationary phase . Quasi-lysis and lethality were not observed . The primary effect of the induction of the kil gene was the overproduction of beta-glucanase . The total amount produced per milliliter of bacterial culture was almost threefold higher than that of the corresponding Kil- control . The protein pattern of periplasm and culture medium was analyzed before and after induction of the kil gene expression, indicating that the release of periplasmic proteins is semiselective . This secretion system is the first to use a growth-phase-regulated promoter for the expression of the kil gene. Arch Microbiol, 1997 Mar 7, 167(2/3), 126 - 36 Requirement of a large K+-uptake capacity and of extracytoplasmic protease activity for protamine resistance of Escherichia coli Stumpe S, Bakker EP. The effect of protamine on growing cells of Escherichia coli K-12 strains containing different K+-uptake systems was investigated . Immediately after the addition of the toxic peptide, growth ceased and all strains lost most of their K+ . In addition, these cells released a significant amount of their ATP into the medium, and the cytoplasmic volume of these cells decreased by 70% . Whereas cells without rapid K+-uptake systems did not recover, cells containing either the Trk systems or the overproduced Kup system slowly reversed the effects of protamine, and growth resumed after the cells had reached their original volume . Experiments with a set of strains carrying mutations in the K+-uptake gene trkA showed a reasonably satisfactory correlation between inhibition of net K+ uptake and the lag time for resumption of growth after addition of protamine . Cells carrying mutations in three extracytoplasmic proteases were hypersusceptible to protamine, suggesting that the toxic peptide is degraded by these proteases . Data on the effect of a second addition of protamine suggest that protamine degradation activity is inducible . These data are interpreted to mean that reaccumulation of K+ by protamine-treated cells triggers recovery of the cells, thereby allowing induction of extracytoplasmic proteases . These, in turn, degrade protamine, leading to complete recovery of the cells and resumption of growth . Cells that cannot take up K+ rapidly remain metabolically compromised to such an extent that extracytoplasmic protease activity is not induced, leading to a prolonged susceptibility of the cells to the toxic peptide. Biochem Biophys Res Commun, 1997 Mar 6, 232(1), 231 - 5 Expression, purification, and partial characterization of HCV RNA polymerase; Yuan ZH et al.; The product of the NS5B gene of Hepatitis C Virus (HCV) has been expressed in Escherichia Coli both as a fusion protein with glutathione-S-transferase (GST) of molecular weight 91 KDa and at high level as a single protein of molecular weight 65 KDa . The protein was sequestered within inclusion bodies and a variety of procedures designed to minimize inclusion body formation proved unsuccessful . The method finally adopted involved the purification of inclusion bodies followed by the solubilization, purification, and refolding of the expressed protein . A good recovery and protein purity of the order of 80-90% were achieved . The purified protein was shown to possess RNA polymerase activity in an assay using polyA/oligoU as template . The enzymatic activity is rifampicin resistant, poly A dependent, and requires Mg++ . The availability of purified HCV RNA polymerase will allow the study of viral replication and constitute the basis for testing new anti-viral drugs. Biochem Biophys Res Commun, 1997 Mar 6, 232(1), 198 - 203 Molecular cloning and expression of cDNAs encoding rat brain and liver cytosolic long-chain acyl-CoA hydrolases; Yamada J et al.; cDNAs encoding the long-chain acyl-CoA hydrolases (ACHs) from rat brain and liver, referred to as rBACH and rLACH1, respectively, were isolated and sequenced . The rBACH cDNA contained an open reading frame encoding a 338-amino acid polypeptide with a calculated molecular weight of 37,559, of which the deduced amino acid sequence matched partial amino acid sequences directly determined for peptides generated by tryptic digestion or CNBr cleavage of purified rBACH . The rLACH1 cDNA contained an open reading frame encoding a 343-amino acid polypeptide with a molecular weight of 38,240 . When expressed in Escherichia coli, these cDNAs produced palmitoyl-CoA hydrolase activity and 44-kDa proteins with molecular masses similar to those of purified rBACH and rLACH1 (43 kDa) . These expressed proteins and enzyme activity were immunoblotted and neutralized, respectively, by anti-rBACH or anti-rLACH1 antibodies . rLACH1 cDNA had 84 and 94% identity with rBACH cDNA at the nucleotide and amino acid levels, respectively . However, the 5'-end of the former cDNA which contained the N-terminal coding region of rLACH1 was entirely different from the corresponding region of rBACH cDNA, suggesting that these enzymes may be generated by alternative use of exons of the same gene . Northern blot analysis showed that ACH mRNA was expressed constitutively in the rat brain and testis, whereas its expression in the liver was inducible by treatment with the peroxisome proliferator . This study demonstrated the molecular diversity of ACH and suggested the presence of tissue-specific mechanisms to regulate the ACH gene expression. Biochem Biophys Res Commun, 1997 Mar 6, 232(1), 130 - 5 Conformational transition of DnaA protein by ATP: structural analysis of DnaA protein, the initiator of Escherichia coli chromosome replication; Kubota T et al.; DnaA protein binds to the chromosomal origin (oriC) to initiate DNA replication . We developed an efficient system for purification of DnaA protein which will facilitate physicochemical analysis of the protein . The yield of DnaA protein was increased at least 6-fold compared to an available method being used, and over 22 mg of the protein were obtained from only 100 g of cells . DnaA protein purified by this procedure showed an indistinguishable affinity for ATP, and activity for in vitro replication of oriC plasmid . The process of denaturation of DnaA protein, which was blocked by ATP, was monitored by intrinsic fluorescence and circular dichroism . Analysis of circular dichroism revealed that DnaA protein is rich in alpha-helices, and that ATP-binding leads to a significant transition of protein conformation in that the content of alpha-helices is decreased . This is the first evidence indicating that ATP-binding profoundly affects conformation of DnaA protein. Nature, 1997 Mar 6, 386(6620), 91 - 4 Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy; Conway JF et al.; Despite the development of vaccines, the hepatitis B virus remains a major cause of human liver disease . The virion consists of a lipoprotein envelope surrounding an icosahedral capsid composed of dimers of a 183-residue protein, 'core antigen' (HBcAg) . Knowledge of its structure is important for the design of antiviral drugs, but it has yet to be determined . Residues 150-183 are known to form a protamine-like domain required for packaging RNA, and residues 1-149 form the 'assembly domain' that polymerizes into capsids and, unusually for a capsid protein, is highly alpha-helical . Density maps calculated from cryo-electron micrographs show that the assembly domain dimer is T-shaped: its stem constitutes the dimer interface and the tips of its arms make the polymerization contacts . By refining the procedures used to calculate the map, we have extended the resolution to 9 A, revealing major elements of secondary structure . In particular, the stem, which protrudes as a spike on the capsid's outer surface, is a 4-helix bundle, formed by the pairing of alpha-helical hairpins from both subunits. Mutat Res, 1997 Mar 4, 374(1), 21 - 40 Statistical analysis of the lacI transgenic mouse mutagenicity assay; Fung KY et al.; The transgenic mouse assay is now widely used to test chemicals for genotoxic potential . In this article, we consider statistical tests for increasing trend in mutant frequency with increasing dose, along with statistical models that may be used to describe the observed dose-response relationships . The application of these methods is illustrated using data on 2-acetylaminofluorene, di(2-ethylhexyl)phthalate, heptachlor, and sodium phenobarbital . No strong evidence of extra-binomial variation was detected at the plate level, but greater evidence was noted when the data were aggregated to the package or animal level in liver, necessitating the use of statistical methods that allow for overdispersion relative to binomial variation . Clear increase on mutant frequency induced by 2-acetylaminofluorene was detected in both liver and bladder, but no apparent trends were noted with di(2-ethylhexyl)phthalate, heptachlor, and sodium phenobarbital . The exponential model provides a good fit to the observed dose-response relationship in liver, whereas a Weibull model provides a better fit for bladder. Biochemistry, 1997 Mar 4, 36(9), 2622 - 36 Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: structural and functional characterization of mutants of Asp 26 and Lys 57; Dyson HJ et al.; To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes . Both the oxidized (Trx-S2) and reduced {Trx-(SH)2} forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt) . The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar Kd values (approximately 5 nM) for both mutants and wt . In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations . In the reaction of Trx-S2 with thioredoxin reductase at pH 8.0, the kcat/Km value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold . The activity of Trx-(SH)2 as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin . At 15 degrees C and pH 8.0, both the D26A and K57M mutants showed 5--10-fold decreases in rates of reaction compared to those of the wild type, and the pH-rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve . NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region . The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pKa of 7.5 in wt Trx-S2 as Asp 26 . A profound change in the pKa of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S2 . The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)2 proteins . A single pKa shifted to higher values is observed on both the Cys 32 and Cys 35 Cbeta resonances . Ultraviolet absorbance measurements (A240) as a function of pH for wt Trx-(SH)2 demonstrate that the cysteine thiols titrate with apparent pK(a)s of about 7.1 and 9.9 . The mutant proteins each show a single transition in the A240 measurements, with a midpoint at pH 7.8-8.0, consistent with the NMR results . The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two . It is clear that both thiol pK(a)s have been significantly shifted by the mutations . The Cys 32 pKa is moved from 7.1 in wt to 7.8-8.0 in the mutants . The value of the Cys 35 pKa either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance measurements or else is shifted to much higher pHs (> 10) where its transition is masked in both UV and NMR measurements by the effects of ionization of the tyrosine residues and unfolding of the protein . Our results strongly suggest that the buried Asp 26 carboxyl and Lys 57 epsilon-amino groups significantly affect the pK(a)s of the active site thiols, particularly that of the exposed low-pKa thiol Cys 32, thereby enhancing the rates of thiol-disulfide reactions at physiological pH. Biochemistry, 1997 Mar 4, 36(9), 2577 - 85 Solution structure of an RNA.2'-O-methylated RNA hybrid duplex containing an RNA.DNA hybrid segment at the center; Nishizaki T et al.; The solution structure of an RNA.2'-O-methylated RNA hybrid duplex containing an RNA.DNA hybrid segment at its center, (ggagaugac).(GmUmCmATCTCmCm), where lowercase letters, capital letters, and capital letters with the subscript m are RNA, DNA, and 2'-O-methylated RNA residues, respectively, was determined by observing the NMR spectra and performing the full relaxation matrix refinement . The 2'-O-methylation gives several characteristic features to oligoribonucleotides . In addition, this hybrid duplex is cleaved at a specific position by Escherichia coli ribonuclease HI, and so the role of the tertiary structure during the substrate recognition by the enzyme is of interest . The NOE connectivities among the proton resonances revealed that the duplex was a right handed helix . The 2'-O-methylated RNA segments had a typical C3'-endo conformation, and the 2'-O-methyl groups were directed to the minor groove of this duplex, taking the torsion angles phi (C1'-C2'-02'-CH3) that were all gauche(+) . The DNA residues in the central RNA.DNA hybrid duplex formed the C3'-endo conformation, except for the middle thymine residue . No remarkable structural discontinuities were observed around the junction sites at either the 5'- or 3'-end of the DNA . The overall structure was close to the typical A-form duplex. Biochemistry, 1997 Mar 4, 36(9), 2544 - 9 Protein dissection of the antiparallel coiled coil from Escherichia coli seryl tRNA synthetase; Oakley MG et al.; The alpha-helices of coiled-coil proteins are predominantly parallel, in contrast to the general preference for an antiparallel orientation of interacting alpha-helices found in globular proteins . One intriguing exception is the antiparallel, two-stranded coiled coil comprising the long helical arm of the bacterial seryl tRNA synthetases (SRS) . A recombinant 82-residue peptide corresponding to the helical arm of Escherichia coli SRS folds into a stable, monomeric, helical structure in the absence of the rest of the protein, as shown by circular dichroism (CD) and equilibrium sedimentation centrifugation . However, peptides corresponding to the individual helices of SRS are unstructured at neutral pH and do not associate appreciably at total peptide concentrations up to 100 microM . Covalent attachment of the the two peptides through a nonnatural, disulfide-containing linker restores structure and allows study of variants in which the individual helices are constrained to interact in either an antiparallel or a parallel orientation . We find that the antiparallel species are substantially more helical and more stable to thermal denaturation than their parallel counterpart . Thus, the SRS helical arm is an autonomously folding unit, and, unlike most other coiled coils, has an intrinsic preference for an antiparallel orientation of its constituent helices. Biochemistry, 1997 Mar 4, 36(9), 2517 - 30 Solution structure of the 30 kDa N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system by multidimensional NMR; Garrett DS et al.; The three-dimensional solution structure of the 259-residue 30 kDa N-terminal domain of enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been determined by multidimensional nuclear magnetic resonance spectroscopy . Enzyme I, which is autophosphorylated by phosphoenolpyruvate, reversibly phosphorylates the phosphocarrier protein HPr, which in turn phosphorylates a group of membrane-associated proteins, known as enzymes II . To facilitate and confirm NH, 15N, and 13C assignments, extensive use was made of perdeuterated 15N- and 15N/13C-labeled protein to narrow line widths . Ninety-eight percent of the 1H, 15N, and 13C assignments for the backbone and first side chain atoms of protonated EIN were obtained using a combination of double and triple resonance correlation experiments . The structure determination was based on a total of 4251 experimental NMR restraints, and the precision of the coordinates for the final 50 simulated annealing structures is 0.79 +/- 0.18 A for the backbone atoms and 1.06 +/- 0.15 A for all atoms . The structure is ellipsoidal in shape, approximately 78 A long and 32 A wide, and comprises two domains: an alpha/beta domain (residues 1-20 and 148-230) consisting of six strands and three helices and an alpha-domain (residues 33-143) consisting of four helices . The two domains are connected by two linkers (residues 21-32 and 144-147), and in addition, at the C-terminus there is another helix which serves as a linker between the N- and C-terminal domains of intact enzyme I . A comparison with the recently solved X-ray structure of EIN {Liao, D.-I., Silverton, E., Seok, Y.-J., Lee, B . R., Peterkofsky, A., & Davies, D . R . (1996) Structure 4, 861-872} indicates that there are no significant differences between the solution and crystal structures within the errors of the coordinates . The active site His189 is located in a cleft at the junction of the alpha and alpha/beta domains and has a pKa of approximately 6.3 . His189 has a trans conformation about chi1, a g+ conformation about chi2, and its Nepsilon2 atom accepts a hydrogen bond from the hydroxyl proton of Thr168 . Since His189 is thought to be phosphorylated at the N epsilon2 position, its side chain conformation would have to change upon phosphorylation. Biochemistry, 1997 Mar 4, 36(9), 2450 - 8 De novo design of native proteins: characterization of proteins intended to fold into antiparallel, rop-like, four-helix bundles; Betz SF et al.; The de novo design and characterization of a series of 51-residue helix-turn-helix peptides intended to dimerize into antiparallel four-stranded coiled coils is described . The sequence is based on a coiled coil heptad repeat Ncap-(Aa Zb Zc Ld Ze Zf Zg)3-turn- (Xa Zb Zc Ld Ze Zf Zg)3-Ccap-CONH2, where X is either Val or Ala . The overall topology was intended to be similar to that found in the Escherichia coli protein ROP . The design strategy included consideration of geometric complementarity of the packing of side chains within the hydrophobic core as well as the use of specific interfacial interactions, both of which were intended to favor the desired ROP-like topology . Additionally, the sequence was designed to destabilize potential alternative structures that might compete with the desired topology . The peptides (RLP-1, RLP-2, and RLP-3) assemble into stable alpha-helical dimers and exhibit the hallmarks of a native protein as judged by its spectroscopic properties, and the lack of binding to hydrophobic dyes . Also, the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence of the CD spectra and confirmed by differential scanning calorimetry (DSC) . The values determined by the two methods are in excellent agreement and are in the range of those of naturally occurring proteins of this size . These results suggest that it is now possible to design native-like helical proteins that should serve as templates for the further design of functional proteins. Biochemistry, 1997 Mar 4, 36(9), 2425 - 38 Ribonuclease P catalysis requires Mg2+ coordinated to the pro-RP oxygen of the scissile bond; Chen Y et al.; Ribonuclease P (RNase P) is an essential enzyme whose action produces the mature 5' termini of all cellular and organellar transfer RNA molecules . In bacteria, the catalytic subunit of RNase P is an RNA molecule which by itself can bind substrate pre-tRNA, select and hydrolyze the correct phosphodiester bond, and release product tRNA . The simple requirements of the reaction-a monovalent cation such as K+ or NH4+ and the divalent cation Mg2+ (or Mn2+)-have prompted proposals that all aspects of phosphodiester bond hydrolysis might be accomplished by one or more divalent metal cations coordinated to the enzyme or substrate . To precisely localize the ligands of catalytically-involved Mg2+, we assayed cleavage by Escherichia coli RNase P RNA of pre-tRNA in which specific pro-Rp phosphate oxygens were replaced with sulfur . RNase P cleavage was targeted to that bond, at or nearest to the normal cleavage site, at which Mg2+ or Mn2+ could be coordinated . Single-turnover kinetics demonstrated that the apparent rate constant for the hydrolysis event was determined quantitatively by the affinity of the divalent cation (Mg2+ or Mn2+) for the atom (O or S) at the pro-Rp position of the scissile phosphodiester bond . We propose a model for pre-tRNA cleavage in which an essential Mg2+ ion is coordinated directly to the pro-Rp phosphate oxygen and indirectly to two other ligands near the scissile bond: the upstream ribose 2'-hydroxyl and the downstream purine N7 . This catalytic Mg2+ ion most likely positions and deprotonates a water molecule for in-line nucleophilic attack on the scissile bond phosphorus. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 2085 - 90 The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose; Bonin CP et al.; GDP-L-fucose is the activated nucleotide sugar form of L-fucose, which is a constituent of many structural polysaccharides and glycoproteins in various organisms . The de novo synthesis of GDP-L-fucose from GDP-D-mannose encompasses three catalytic steps, a 4,6-dehydration, a 3,5-epimerization, and a 4-reduction . The mur1 mutant of Arabidopsis is deficient in L-fucose in the shoot and is rescued by growth in the presence of exogenously supplied L-fucose . Biochemical assays of the de novo pathway for the synthesis of GDP-L-fucose indicated that mur1 was blocked in the first nucleotide sugar interconversion step, a GDP-D-mannose-4,6-dehydratase . An expressed sequence tag was identified that showed significant sequence similarity to proposed bacterial GDP-D-mannose-4,6-dehydratases and was tightly linked to the mur1 locus . A full-length clone was isolated from a cDNA library, and its coding region was expressed in Escherichia coli . The recombinant protein exhibited GDP-D-mannose-4,6-dehydratase activity in vitro and was able to complement mur1 extracts in vitro to complete the pathway for the synthesis of GDP-L-fucose . All seven mur1 alleles investigated showed single point mutations in the coding region for the 4,6-dehydratase, confirming that it represents the MUR1 gene. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1761 - 6 Site-specific mutagenesis reveals differences in the structural bases for tight binding of RNase inhibitor to angiogenin and RNase A; Chen CZ et al.; RNase inhibitor (RI) binds with extraordinary affinity (Ki approximately 10(-13)-10(-16) M) to diverse proteins in the pancreatic RNase superfamily . In the present study, the structural basis for the recognition of two RI ligands, human angiogenin (Ang) and bovine RNase A, has been investigated by site-specific mutagenesis of human RI and Ang . The RI residues examined were those that appear to form strong contacts with RNase A in the crystal structure of the porcine RI x RNase A complex {Kobe, B . & Deisenhofer, J . (1995) Nature (London) 374, 183-186} that would not be replicated in the Ang complex . Ala substitutions of five of these residues (Glu-287, Lys-320, Glu-401, Cys-408, and Arg-457) were found to have little or no effect on binding of RNase A . In contrast, replacements of Tyr-434, Asp-435, and Tyr-437 and deletion of the C-terminal residue Ser-460 substantially weakened affinity for RNase A: the losses of binding energy associated with the mutations were 5.9, 3.6, 2.6, and 3.5 kcal/mol, respectively . Thus these four residues, which are neighbors in the tertiary structure, appear to constitute a "hot spot" for the RNase A interaction . However, only one of them, Asp-435, was equally important for binding of Ang; the Ki increases produced by mutations of the others were 20- to 93-fold smaller for Ang than for RNase A . Consequently, Tyr-434 plays a significant but lesser role in the Ang complex, whereas Tyr-437 and Ser-460 make only minor contributions . Ala mutations of four Ang residues (His-8, Gln-12, Asn-68, and Glu-108) that correspond to RI contacts on RNase A produced no major changes in affinity for RI . These findings indicate that RI uses largely different interactions to achieve its extremely tight binding of RNase A and Ang. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1755 - 60 Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded; Komissarova N et al.; RNA polymerase (RNAP) may become arrested during transcript elongation when ternary complexes remain intact but further RNA synthesis is blocked . Using a combination of DNA and RNA footprinting techniques, we demonstrate that the loss of catalytic activity upon arrest of Escherichia coli RNAP is accompanied by an isomerization of the ternary complex in which the enzyme disengages from the 3' end of the transcript and moves backward along the DNA with concomitant reverse threading of the intact RNA through the enzyme . The reversal of RNAP brings the active center to the internal RNA position and thereby it represents a step in factor-facilitated transcript cleavage . Secondary structure elements or the 5' end of the transcript can prevent the isomerization by blocking the RNA threading . The described novel property of RNAP has far-reaching implications for the understanding of the elongation mechanism and gene regulation. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1745 - 8 Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor; Meacci E et al.; Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the Saccharomyces cerevisiae Sec7 gene product, which is crucial in protein transport . More recently, the same protein has been reported to be a regulatory factor for the alphaLbeta2 integrin in lymphocytes . Overexpression of human or yeast ADP-ribosylation factor (ARF) genes rescues yeast with Sec7 defects, restoring secretory pathway function . ARFs, 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and now recognized as critical components in intracellular vesicular transport, exist in an inactive cytosolic form with GDP bound (ARF-GDP) . Interaction with a guanine nucleotide-exchange protein (GEP) accelerates exchange of GDP for GTP, producing the active ARF-GTP . Both soluble and particulate GEPs have been described . To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in Escherichia coli, on ARF activity were evaluated . Cytohesin-1 enhanced binding of 35S-labeled guanosine 5'-{gamma-thio}triphosphate {35S}GTP{gammaS} or {3H}GDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP) . Addition of cytohesin-1 to ARF3 with {35S}GTP{gammaS} bound, accelerated {35S}GTP{gammaS} release to a similar degree in the presence of unlabeled GDP or GTP{gammaS} and to a lesser degree with GDP{betaS}; release was negligible without added nucleotide . Cytohesin-1 also increased ARF1 binding to a Golgi fraction, but its effect was not inhibited by brefeldin A (BFA), a drug that reversibly inhibits Golgi function . In this regard, it differs from a recently reported BFA-sensitive ARF-GEP that contains a Sec7 domain. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1733 - 8 Cloning of murine RNA polymerase I-specific TAF factors: conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1; Heix J et al.; Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs) . Unlike class II- and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter . As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts . The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex . Thus, primate vs . rodent promoter selectivity mediated by the TBP-TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1709 - 14 The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element; Murakami K et al.; RNA polymerase core enzyme of Escherichia coli is composed of two alpha subunits and one each of the beta and beta' subunits . The C-terminal domain of the RNA polymerase alpha subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface . To identify possible differences in the functional roles of the two alpha subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct alpha subunit derivatives in a defined orientation ("oriented alpha-heterodimer") . The binding sites of two alpha C-terminal domains on the UP element DNA were determined by hydroxyl radical-based DNA cleavage mediated by (p-bromoacetamidobenzyl)-EDTA x Fe, which was bound at Cys-269 on the UP recognition surface of one or both alpha subunits . The results clearly indicated that the two alpha subunits bind in tandem to two helix turns of the rrnBP1 UP element, and that the beta'-associated alpha subunit is bound to the promoter-distal region. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1663 - 8 "Peptabody": a new type of high avidity binding protein; Terskikh AV et al.; A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction . A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule . In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library . A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain . The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography . Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself . Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds . Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity . This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1651 - 6 Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo; Oudrhiri N et al.; Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis . Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo . The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture . Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo . Transfected cells were detected both in the surface epithelium and in submucosal glands . In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system. Virology, 1997 Mar 3, 229(1), 279 - 82 Demonstration of equine herpesvirus-1 neuronal latency in murine olfactory bulbs using a novel combined in situ PCR and protein synthesis method; Marshall KR et al.; Equine herpesvirus-1 (EHV-1) latency in murine olfactory bulbs was demonstrated by a novel combined in situ PCR and in vitro protein synthesis method (in situ PS-PCR) . The Escherichia coli lacZ gene replacing a deletion in EHV-1 gene 71 (EUS4) was thus amplified and transcribed/translated in situ followed by enzymatic detection using X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside) . beta-Galactosidase was found to be concentrated over mitral/tufted neurons indicating those to be the sites of latency . Our results suggest that, in common with other alpha-herpesviruses, EHV-1 can establish latency in central nervous system neurons and that the unique membrane glycoprotein encoded by EHV-1 gene 71 is nonessential for infection of neural tissues. Virology, 1997 Mar 3, 229(1), 201 - 11 Characterization of the African swine fever virus structural protein p14.5: a DNA binding protein; Martinez-Pomares L et al.; The gene encoding the structural protein p14.5 of African swine fever virus (ASFV) has been mapped and sequenced . This gene, designated E120R, is located in the Sa/l H/EcoRl E restriction fragment of the ASFV genome and is predicted to encode a protein of 120 amino acids with a molecular weight of 13.4 kDa . Northern-blot analysis showed that E120R is transcribed at late times during the viral replication cycle . The E120R gene product has been expressed in Escherichia coli, purified, and used as an antigen for antibody production . The antiserum anti-pE120R recognized a protein in infected cell extracts with an apparent molecular mass of 14.5 kDa, named p14.5 . This antiserum also detected protein p14.5 in purified virus particles . Protein p14.5 is synthesized late in infection and is located in viral factories . Immunoprecipitation analysis and binding-assay experiments have shown that protein p14.5 interacts with a protein that could correspond to the major structural protein p72 . Purified protein p14.5 interacts with DNA in a sequence-independent manner . It binds to both single-stranded and double-stranded DNA . A possible role of protein p14.5 in the encapsidation of ASFV DNA is suggested. EMBO J, 1997 Mar 3, 16(5), 989 - 97 1.9 A crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling; Somers W et al.; Interleukin 6 (IL-6) has many biological activities in vivo, and deregulation has been implicated in many disease processes . IL-6, a 185 amino acid polypeptide was refolded, purified and crystallized . The crystals diffracted to beyond 1.9 A and the structure was solved using single isomorphous replacement . The X-ray structure of IL-6 is composed of a four helix bundle linked by loops and an additional mini-helix . 157 out of 185 residues are well defined in the final structure, with 18 N-terminal and 8 A-B loop amino acids displaying no interpretable electron density . The three-dimensional structure has been used to construct a model of IL-6 interacting with the IL-6 receptor (alpha-chain) and gp130 (beta-chain) that gives new insight into the process of molecular recognition and signaling . Based on this model, we predict a fourth binding site on IL-6, a low affinity IL-6-IL-6 interaction, which may be necessary for the sequential assembly of a functional hexameric IL-6 receptor complex. FEBS Lett, 1997 Mar 3, 404(1), 65 - 9 Rab proteins of Drosophila melanogaster: novel members of the Rab-protein family; Satoh AK et al.; From a Drosophila head cDNA library, we isolated 9 cDNA clones, each of which encodes a different member of Rab-protein family . Seven of them (DRabs) have more than 80% amino acid identity to the corresponding members of mammalian Rab proteins . The other two proteins (DRabRP3 and 4) show low sequence similarity to any of the known Rab proteins . However, both contain all amino acids conserved in known Rabs . In addition, DRabRP4 has strong GTP-binding activity, when synthesized in E . coli cells . These results indicate that DRabRPs are novel members of the Rab-protein family . Molecular phylogenetic analysis also supported this conclusion. FEBS Lett, 1997 Mar 3, 404(1), 45 - 50 Secondary structure of the IIB domain of the Escherichia coli mannose transporter, a new fold in the class of alpha/beta twisted open-sheet structures; Gschwind RM et al.; The mannose transporter of the Escherichia coli bacterial phosphotransferase system consists of three subunits: IIAB, IIC and IID . IIABMan transfers phosphoryl groups to the transported substrate via phosphohistidine intermediates . Its IIB domain was overexpressed and isotopically labelled with 13C, 15N and 2H . Heteronuclear 3D triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances . Based on the evaluation of conformationally sensitive parameters, the secondary structure of the IIBMan domain has been determined as an alpha/beta twisted open-sheet structure consisting of a six-stranded parallel beta-sheet with the novel strand order 3-2-4-1-5-6, six helices and a short two-stranded antiparallel beta-sheet. FEBS Lett, 1997 Mar 3, 404(1), 15 - 8 Nucleotide occupancy of F1-ATPase catalytic sites under crystallization conditions; Lobau S et al.; Using site-directed tryptophan fluorescence we studied nucleotide occupancy of the catalytic sites of Escherichia coli F1-ATPase, under conditions used previously for crystallization and X-ray structure analysis of the bovine mitochondrial enzyme {Abrahams et al . (1994) Nature 370, 621-628} . We found that only two of the three catalytic sites were filled in the E . coli enzyme under these conditions (250 microM MgAMPPNP plus 5 microM MgADP), consistent with what was reported in the bovine F1 X-ray structure . However, subsequent addition of a physiological concentration of MgATP readily filled the third catalytic site . Therefore the enzyme form seen in the X-ray structure results from the fact that it is obtained under sub-saturating nucleotide conditions . The data show that the X-ray structure is compatible with a catalytic mechanism in which all three F1-ATPase catalytic sites must fill with MgATP to initiate steady-state hydrolysis {e.g . Weber and Senior (1996) Biochim . Biophys . Acta 1275, 101-104} . The data further demonstrate that the site-directed tryptophan fluorescence technique can provide valuable support for F1 crystallography studies. Cytokines Cell Mol Ther, 1997 Mar, 3(1), 27 - 32 Incidence of antibodies to interferon-beta in patients treated with recombinant human interferon-beta 1a from mammalian cells; Abdul-Ahad AK et al.; Patients receiving recombinant human interferon-beta 1a (IFN-beta 1a) produced in Chinese hamster ovary (CHO) cells were tested for the formation of neutralizing antibodies (NABs) to IFN-beta . Samples were tested in an enzyme-linked immunosorbent assay (ELISA), and if positive were then tested for neutralization of antiviral activity in an IFN-beta bioassay . A total of 793 patients with viral diseases, premalignant and malignant diseases, and multiple sclerosis received IFN-beta 1a in clinical studies . Long-term studies included 56 patients with cancer treated for 6 or 12 months and 334 patients with multiple sclerosis (MS) at the end of one year of treatment . All of the NAB-positive patients were found in the latter . Positivity in a single specimen was found in 14.4% of the MS patients . The incidence of sustained neutralizing antibody titres (i.e . positive in two tests at least 6 months apart) was 6.9% in this group . Comparison with results from other studies suggests that CHO-derived IFN-beta 1a induces less neutralizing antibody than IFN-beta 1b produced in E . coli. Biochemistry (Mosc), 1997 Mar, 62(3), 337 - 41 Stability and stabilization of recombinant peroxidase in reversed micelles; Klyachko NL et al.; Stability of recombinant peroxidase lacking carbohydrate residues on the surface of the protein molecule has been characterized in reversed micelles of Aerosol OT in octane . The enzyme stability was found to depend on the surfactant hydration degree (w0 = {H2O}/{AOT}) . Residual activity after 1 h incubation dropped to zero at w0 = 7 but was 54% at w0 = 25 . However, the residual activity levels at all values of hydration degree were definitely low compared to that of glycosylated wild-type horseradish peroxidase . The stability of the enzyme apparently depends on the presence of carbohydrate residues . Stabilization of recombinant peroxidase in reversed micellar system involved sugar-containing co-surfactants such as Tweens and Spans is proposed . As an example, addition of 1 mM Span 80 (1% relative to AOT concentration) increased the recombinant peroxidase stability up to that of wild-type peroxidase. Biochemistry (Mosc), 1997 Mar, 62(3), 233 - 6 A hybrid mutant form of Escherichia coli inorganic pyrophosphatase; Velichko IS et al.; The inorganic pyrophosphatase of Escherichia coli is a tightly hexamer of identical subunits . Upon interaction of its two mutant forms in which the trimer-trimer contacts were weakened because of E20D and H136Q substitutions, a hybrid hexameric E20D/H136Q-PPase is formed . The catalytic activity of its constituent H136Q trimer is same of its hexamer, whereas metal-binding affinity is significantly decreased . These results point to an interdependence of two trimers in catalysis by hexameric pyrophosphatase. J Magn Reson, 1997 Mar, 125(1), 34 - 42 Protein heteronuclear NMR assignments using mean-field simulated annealing; Buchler NE et al.; A computational method for the assignment of the NMR spectra of larger (21 kDa) proteins using a set of six of the most sensitive heteronuclear multidimensional nuclear magnetic resonance experiments is described . Connectivity data obtained from HNC alpha, HN(CO)C alpha, HN(C alpha)H alpha, and H alpha (C alpha CO)NH and spin-system identification data obtained from CP-(H)CCH-TOCSY and CP-(H)C(C alpha CO)NH-TOCSY were used to perform sequence-specific assignments using a mean-field formalism and simulated annealing . This mean-field method reports the resonance assignments in a probabilistic fashion, displaying the certainty of assignments in an unambiguous and quantitative manner . This technique was applied to the NMR data of the 172-residue peptide-binding domain of the E . coli heat-shock protein, DnaK . The method is demonstrated to be robust to significant amounts of missing, spurious, noisy, extraneous, and erroneous data. Mikrobiologiia, 1997 Mar-Apr, 66(2), 179 - 84 {Release of protein into the extracellular space as a nonspecific response to stress in Escherichia coli}; Roshchina EK et al.; This paper is concerned with the kinetics of excretion of fluorescent tryptophan-containing proteins from Escherichia coli cells kept in physiological saline at room temperature or incubated at 42, 48, and 55 degrees C . The kinetic curves of the extracellular concentration of protein can be described by parameters T and C, where C is the stationary extracellular concentration of the protein and T is the time in which the given concentration is reached . T was found to be a variable, and C was a constant independent of the type and strength of the stress . During the protein release, the viability of the cells was maintained at the initial level, but, after the concentration of the protein reached a stationary value, the culture cells died exponentially . All this allows the protein release into extracellular medium to be considered as a nonspecific response of E . coli to stress . The protein excretion was analyzed with reference to the data on the kinetics of release of other UV-absorbing compounds from the cells. Int J Biochem Cell Biol, 1997 Mar, 29(3), 485 - 91 Recombinant rat liver S-adenosyl-L-methionine synthetase tetramers and dimers are in equilibrium; Mingorance J et al.; Rat liver S-adenosyl-L-methionine synthetase is present in two oligomeric forms, tetramers and dimers, with different substrate kinetics and regulation . In vivo the relative amounts of both forms may change in some instances . The basis of this regulatory mechanism is not known . When rat liver cDNA was used to express the protein in Escherichia coli the two oligomeric forms were found . Gel filtration chromatography of the purified recombinant enzyme suggested that these two isoforms might be in equilibrium . This was confirmed by kinetic experiments which showed that the specific activity of the enzyme was dependent on the protein concentration . From these experiments, apparent equilibrium constants of (5.6 +/- 0.4) x 10(5) M-1 and (3.5 +/- 0.9) x 10(5) M-1 were obtained at 2mM and 60 microM methionine concentrations, respectively . Using hydrophobic chromatography on phenyl-Sepharose to separate the tetrameric and dimeric forms, an equilibrium constant of (4.9 +/- 0.7) x 10(5) M-1 was calculated . A rate constant for the dissociation of the tetramer of k-1 = (8.1 +/- 0.4) x 10(-4) s-1 at 4 degrees C was also calculated using the same approach . In summary, we have shown that the rat liver S-adenosyl-L-methionine synthetase produced in bacterial cells is present in two oligomeric forms, tetramers and dimers, which are in equilibrium . This system might be useful for studying the dynamics and the regulation of the distribution of oligomeric forms in the mammalian liver. Biol Trace Elem Res, 1997 Mar, 56(3), 295 - 309 Enhancement of adriamycin toxicity by iron chelates is not a free radical mechanism; Gelvan D; The possible involvement of metal ions and free radicals in the cytotoxic mechanism of Adriamycin (ADR) was investigated, using a model system of Escherichia coli cells . It is shown that E . coli mediated the production of free radicals under anaerobic (ADR-semiquinone) and aerobic (superoxide) conditions . ADR-induced loss of colony-forming ability was enhanced by the addition of iron (Fe) chelates . These observations suggested that a Fenton-type free radical mechanism was responsible for ADR toxicity . However, the mortality rate was essentially unchanged by the exclusion of oxygen . It was also unaffected by the addition of H2O2, catalase, or chelating agents . Cu(II), Zn(II) or Mg(II) had no effect on ADR toxicity . ADR and iron chelates did not induce measurable amounts of DNA strand-breaks . These observations suggest a mechanism of ADR-induced cell killing that is enhanced by Fe chelates, but does not directly involve oxygen-derived free radicals. Bioorg Khim, 1997 Mar, 23(3), 200 - 4 {Regulation of translation of the distal lacZ gene in polycistronic mRNA by the ribosome stream from the proximal gene}; Nikolenko GN et al.; Four series of plasmids (pNSI, pNSII, pNLI, and pNLII) with artificial polycistrons containing the lacZ test gene were constructed . These plasmids coded for polycistronic mRNAs with two different types of cistron (orfZ and lacZ) coupling: in pNSI and pNLI, the orfZ termination codon and the lacZ initiation codon overlapped (type I); in pNSII and pNLII, the orfZ termination codon, was located upstream of the lacZ SD sequence . The length of the orfZ cistron was 60 bp in pNSI and pNSII or 300 bp in pNLI and pNLII . Plasmids with the same type of cistron coupling contained the same lacZ translation initiation region, whereas the structure of the orfZ translation initiation region varied, thereby providing varying efficiency of the orfZ gene translation . The effect of these variations on the efficiency of the lacZ gene translation was evaluated by direct measurement of the beta-galactosidase activity in Escherichia coli cells transformed with the corresponding plasmids . We found that the level of translation of the distal lacZ gene depended on the ribosome stream from the proximal gene and was maximal at the optimal ribosome stream level, which, in turn, depended on the type of cistron coupling. Bioorg Khim, 1997 Mar, 23(3), 183 - 90 {Cloning and expression of cDNA for tobacco rab1, and structural-functional analysis of the protein}; Andreeva AV et al.; rab1 cDNA coding for a small GTP binding protein Rab1 was isolated from cDNA library of tobacco (Nicotiana tabacum) leaves . The primary structure of this protein was deduced from the rab1 structure . Tobacco rab1 cDNA was expressed in Escherichia coli, and the product was purified and shown to exhibit GTPase activity . A set of Rab1 mutants with altered GTP binding and/or GTPase activities was obtained . Polyclonal antipeptide antibodies were raised against a sequence in the C-terminal region of the tobacco Rab1 capable of recognizing this protein. C R Acad Sci III, 1997 Mar, 320(3), 207 - 14 Human deoxycytidine kinase as a conditional mutator in Escherichia coli; Bouzon M et al.; The chemical diversification of DNA precursors was undertaken in Escherichia coli by expressing the human gene for deoxycytidine kinase, and supplying such recombinant strains with nucleoside analogues bearing an altered base or sugar . Arabinocytidine and dideoxycytidine thus became highly toxic to E . coli in the sub-millimolar range . Deoxynucleosides bearing isoadenine (2-aminopurine) and isoguanine (2-hydroxy-6-aminopurine) showed a high mutagenic potency towards the recombinant strains, to an extent comparable to that of the most efficient mutator alleles (dnaQ) . These findings open the way to the propagation of chemically remodelled nucleic acids and to the controlled hypermutagenesis of plasmids in vivo. Biol Chem, 1997 Mar-Apr, 378(3-4), 321 - 6 Mode of action of cystathionine beta-lyase; Clausen T et al.; Cystathionine beta-lyase (CBL) is a member of the gamma-family of pyridoxal-5'-phosphate (PLP)-dependent enzymes (Alexander et al., 1994) that cleave C(beta,gamma)-S bonds of a broad variety of substrates . Recently, we reported the X-ray crystal structures of CBL and the CBL-trifluoroalanine inactivation complex at 1.83 A and 2.3 A resolution, respectively . The structures explicitly reveal the cofactor and substrate binding pockets . Spectral analysis of substrate turnover indicates a change of hydrophobicity in the microenvironment of the aldimine bond . In combination with further spectroscopic data, crystallographic evidence permits the formulation of a likely reaction mechanism. Biol Chem, 1997 Mar-Apr, 378(3-4), 131 - 40 New insights into the mechanisms and importance of the proteasome in intracellular protein degradation; Goldberg AL et al.; Recent studies of the 20S proteasome from Thermoplasma acidophilum have uncovered some fundamental new properties of its catalytic mechanism . Unlike conventional proteases, 20S and 26S proteasomes degrade protein substrates in a highly processive fashion . They cleave a protein substrate to small peptides before attacking another substrate molecule . This processive behavior is an inherent feature of the 20S particle not requiring cofactors or ATP hydrolysis . Recently, we have described a proteasome-like particle, HslVU, in Escherichia coli . HslVU is a two-component ATP-dependent protease composed of the proteasome-related peptidase HslV (beta-subunit) and the ATPase HslU . In active HslVU complex, cleavage of small peptides and proteins requires the presence of ATP . EM analysis revealed that HslV and HslU are both ring-shaped particles and that the active HslVU complex is a cylindrical four-ring structure, composed of HslV, a two-ring dodecamer, sandwiched between HslU rings . Elucidation of its mode of action may help us understand the role of ATP in function of the 26S proteasome . Several proteasome-specific inhibitors have been recently identified which block the function of proteasome in vivo . These agents have proven very useful to clarify the intracellular function of the proteasome . In mammalian cells, both the rapid degradation of short-lived regulatory proteins and of abnormal polypeptides and the slower degradation of long-lived proteins are blocked by these agents . Thus, in mammalian cells, the proteasome is the site for the degradation of most cell proteins . In contrast, in budding yeast, proteasome inhibitors block the degradation of short-lived proteins but not the breakdown of long-lived proteins, which can be blocked by inhibitors of vacuolar proteases . The inhibition of proteasome function in yeast and mammalian cells, presumably by causing an accumulation of unfolded proteins, triggers the expression of heat shock proteins and concomitantly increases cell resistance to high temperature and various toxic insults. J Radiat Res (Tokyo), 1997 Mar, 38(1), 37 - 43 Effects of 60Co gamma-rays, ultraviolet light, and mitomycin C on Halobacterium salinarium and Thiobacillus intermedius; Shahmohammadi HR et al.; Lethal effects of 60Co gamma-rays, UV light, and mitomycin C on two kinds of bacteria, Halobacterium salinarium which grows in highly concentrated salt media and Thiobacillus intermedius which requires reduced sulfur compounds, were studied and compared with those on Escherichia coli B/r . D37 values for H . salinarium, T . intermedius and E . coli B/r were 393, 150, and 92 Gy, respectively, by exposure to 60Co gamma-rays . They were 212, 38, and 10 J/m2, respectively, by exposure to UV light and 2.36, 0.25, and 0.53 microgram/ml/h, respectively, by exposure to mitomycin C . Against these agents, H . salinarium was much more resistant than T . intermedius and E . coli B/r. J Radiat Res (Tokyo), 1997 Mar, 38(1), 27 - 36 Spectrum of spontaneous mutations in the cyclic AMP receptor protein gene on chromosomal DNA of Escherichia coli; Takimoto K et al.; We determined 46 spontaneous mutations occurring in the cyclic AMP receptor protein gene (crp) on the chromosomal DNA of Escherichia coli by the use of PCR cloning . Of 24 base substitutions, 17 were transversions and 7 transitions including all types of base substitutions . The frequency of the changes of A:T base pairs was similar to that of G:C pairs, suggesting that A:T pair is also a target for base substitution . Frameshifts including seven-1 and four +1 frameshifts occurred at the sites of a run of identical bases . Deletions extending 18 and 172 bases occurred at the sites where the deleted sequences were flanked by short repeated sequences at the junction . The insertions of IS2 element or its inverted sequence were detected in two and six mutations, respectively . The assay system of the mutation used here is available for the determination of the mutational spectrum of base substitutions. Immunotechnology, 1997 Mar, 3(1), 45 - 59 Humanization of an antibody recognizing a breast cancer specific epitope by CDR-grafting; Fiorentini S et al.; BACKGROUND: Muc1-H23 is a cell surface mucin that is expressed on normal breast luminal epithelial cells and over-expressed in most breast tumors . In addition, Muc-1 expressed by malignant cells is glycosylated differently than Muc-1 expressed by normal cells . This difference in glycosylation exposes a peptide epitope on malignant cells which is not exposed on normal cells . Murine monoclonal antibody H23 recognizes this epitope and stains 91% of breast cancers, but only 1/56 non-malignant breast tissue samples . OBJECTIVE: To create a human antibody that was equivalent to H23 for potential uses in imaging and/or the therapy of breast cancer . STUDY DESIGN: We decided to humanize H23 by CDR-grafting using overlap PCR, and to this end, designed and constructed a bacterial expression vector that would allow V-regions, cloned via unique restriction sites, to be expressed as Fab fragments . In this way, we hoped to be able to rapidly evaluate Fab constructs for binding to Muc-1 and to cells and tissue sections that expressed the antigen . RESULTS: A fully humanized Fab fragment was able to bind Muc-1 peptide, as well as breast cancer cells known to express the epitope and tissue sections, generally showing the same reactivity as the native antibody . In addition, an analysis of sFab expressed with a {His}6 tag preceded by a factor Xa proteolytic cleavage site suggested that E . coli periplasmic signal peptidase was able to cleave the factor Xa site, thereby removing the {His}6 tag . CONCLUSION: We have generated a human antibody that is capable of recognizing a tumor specific epitope expressed by 91% of breast cancers. Protein Eng, 1997 Mar, 10(3), 217 - 22 Prediction of the biologically active sites in eclosion hormone from the silkworm, Bombyx mori; Kikuchi T et al.; The structure-activity relationship of eclosion hormone from the silkworm, Bombyx mori, was analyzed . First, the probable active residues in silkworm eclosion hormone and also tobacco hornworm eclosion hormone were predicted by the average distance map method . To examine the contributions of those residues to the activity of silkworm eclosion hormone, Gly-substituted mutants for those predicted residues were produced by site-directed mutagenesis and their activities were evaluated by a bioassay . Finally, Glu12, Met24 and Phe25 were estimated to be the crucial residues for the eclosion hormone activity . The possibility of the development of a blocker of an eclosion hormone receptor on the basis of the present work is also discussed. Protein Eng, 1997 Mar, 10(3), 285 - 90 Expression and epitope tagging of the membrane anchor subunit (DmsC) of Escherichia coli dimethyl sulfoxide reductase; Turner RJ et al.; Escherichia coli dimethyl sulfoxide reductase is a heterotrimer comprising a catalytic subunit (DmsA), an electron transfer subunit (DmsB) and an integral membrane anchor subunit (DmsC) . DmsC is not antigenic and the production of antibodies to this subunit has not been successful . We have tagged DmsC at the C-terminus with a dystrophin-specific amino acid sequence (dysp) to which antibodies are readily available . We were able to use this tagging technique to monitor expression and localization of DmsC in E . coli and non-muscle eukaryotic cells . Growth properties of wild-type E . coli, strain HB101, overexpressing DmsC:dysp suggest that the expression of DmsC is lethal to E . coli . The lethality could be overcome by utilizing an E . coli F0F1 ATPase mutant as the host . Growth conditions of culture density, duration of induction, temperature of incubation after induction and media conditions were investigated to optimize DmsC:dysp accumulation levels . In order to alleviate the problem arising from the toxicity of DmsC, expression in eukaryotic tissue culture was also explored . A plasmid expressing DmsC:dysp was transfected into COS-1 or McA-RH777 cells . The presence of expressed DmsC:dysp was confirmed using specific anti-dysp antibodies and immunofluorescence microscopy analysis revealed that the DmsC:dysp was localized to the endoplasmic reticulum . Expression of DmsC:dysp did not appear to be toxic to the eukaryotic cells . These data suggest methodologies to overcome lethality problems associated with the overexpression of integral membrane proteins like DmsC. Protein Eng, 1997 Mar, 10(3), 279 - 83 Effects of non-conservative changes to tyrosine 76, a key DNA binding residue of DNase I, on phosphodiester bond cleavage and DNA hydrolysis selectivity; Warren MA et al.; Non-conservative changes, consisting of Y76E, Y76L, Y76Q and Y76W, have been made to tyrosine 76, one of the key DNA binding residues in DNase I . Normally Y76 inserts into the minor groove of DNA and makes an unusual, hydrophobic, stacking interaction with one of the sugars . All four mutants bind to DNA more tightly than the wild type, but cut it more slowly as assessed by Kunitz assays . This gives a rather small decrease in the specificity constants (Vmax/K(m)) for the hydrolysis of DNA, which is roughly paralleled by the loss of activity towards the non-DNA small chromophoric substrate, thymidine-3',5'-di(p-nitrophenyl)phosphate . These non-conservative mutants, therefore, show different behaviour to Y76A and Y76G, studied previously {Doherty A.J., Worrall A.F . and Connolly B.A . (1995) J: Mol . Biol., 251, 366-377} . These two mutants both bind to and cut DNA poorly, resulting in large decreases in Vmax/K(m) values . However, they showed little reduction in rates with the chromophoric substrate . It is likely that the altered side chains in the non-conservative mutants are still able to interact productively with the DNA and contribute to the observed DNA distortion that is essential for efficient catalysis . However, these mutations disrupt the active site, most probably by interference with the hydrogen bonded Y76-E78-H134 triad . H134 is a critical hydrolytic residue of DNase I that is essential for catalysis . The DNA cleavage selectivity of the Y76E, Y76L, Y76Q and Y76W mutants were little altered as compared with the wild-type enzyme as measured using the cutting patterns of a 160 base-pair Escherichia coli Tyr T promoter DNA fragment . This confirms earlier observations, with Y76F, Y76A and Y76G, that showed that this tyrosine has little role in DNA cleavage specificity. Protein Eng, 1997 Mar, 10(3), 263 - 72 Effects of insertions and deletions in a beta-bulge region of Escherichia coli dihydrofolate reductase; Dion-Schultz A et al.; The role of a beta-bulge in Escherichia coli dihydrofolate reductase (DHFR) has been explored by a series of insertion and deletion mutations . Insertion of a seven amino acid sequence from a structurally equivalent 'beta-blowout' sequence from human DHFR destabilizes E . coli DHFR by 3.6 kcal/mol and decreases catalytic efficiency (kcat/K(m)) 34-fold . Deletion of F137, delta 137, the looped out residue in the bulge, also destabilizes E . coli DHFR by 2.8 kcal/mol but only decreases catalytic efficiency threefold . Concurrent deletion of F137 and mutation of, V136 to proline to try and maintain the strand twist associated with the beta-bulge decreases protein stability by 3.4 kcal/mol and decreases catalytic efficiency 84-fold . These insertion/deletion mutations were also constructed in a D27S DHFR background . The D27S mutation has been described previously and proposed to remove the catalytic acid from the active site . The delta 137 mutation partially suppresses the effect of the D27S mutation as it decreases the K(m) for substrate, dihydrofolate, twofold . Non-additive effects are observed for the insertion/deletion mutations in wild-type versus D27S DHFR backgrounds, consistent with structural changes. Electrophoresis, 1997 Mar-Apr, 18(3-4), 588 - 98 Two-dimensional electrophoretic analysis of human breast carcinoma proteins: mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2; Rasmussen RK et al.; MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al . (Eur . J . Biochem . 1993, 213, 701-710), comprises several distinct structural domains including an src homology-3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif . Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction . To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23-77), fused to glutathione S-transferase . This fusion protein was expressed in Escherichia coli, purified using gluthathione-Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2-SH3 domain binding proteins from lysates of 35S-labelled MDA-MB231 human breast tumour cells . Electrophoretic analysis of bound proteins revealed that two low-abundance proteins with a molecular weights (Mr) of approximately 31,500 and approximately 34,000, bound consistently to the MLK2N protein . To establish accurately the Mt / isoelectric point (pI) loci of these MLK2-SH3 domain binding proteins, a number of abundant proteins in a two-dimensional electrophoresis (2-DE) master gel were identified to serve as triangulation marker points . Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in-gel proteolysis and fractionation by rapid (approximately 12 min) microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide-mass fingerprinting and electrospray (ESI)-mass spectrometry (MS)-MS utilizing capillary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analysis . Using this information, a preliminary 2-DE protein database for the human breast carcinoma cell line MDA-MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: l). Electrophoresis, 1997 Mar-Apr, 18(3-4), 498 - 501 Large-scale protein modelling and integration with the SWISS-PROT and SWISS-2DPAGE databases: the example of Escherichia coli; Peitsch MC et al.; Knowledge-based molecular modelling of proteins has proven useful in many instances, including the rational design of mutagenesis experiments, but it has generally been limited by the availability of expensive computer hardware and software . To overcome these limitations, we developed the SWISS-MODEL server for automated knowledge-based protein modelling . The SWISS-MODEL server uses the Brookhaven Protein Data Bank as a source of structural information and automatically generates protein models for sequences which share significant similarities with at least one protein of known three-dimensional structure . We have now used the software framework of the server to generate large collections of protein models, and established the SWISS-MODEL Repository, a new database for automatically generated and theoretical protein models . This repository is directly integrated with the SWISS-PROT and SWISS-2DPAGE databases through the ExPASy World Wide Web server (URL is Here we present an illustration of this process by an application to the Escherichia coli sequences. Vaccine, 1997 Mar, 15(4), 423 - 32 Subgroup specific protection of mice from respiratory syncytial virus infection with peptides encompassing the amino acid region 174-187 from the G glycoprotein: the role of cysteinyl residues in protection; Simard C et al.; We identified subgroup specific protective epitopes represented by the amino acid regions 174-187 and 171-187 of the G glycoproteins from respiratory syncytial virus (RSV), subgroups A and B . Mice immunized with coupled synthetic peptides corresponding to either the region 174-187 containing a Cys186-->Ser substitution or to the native region 171-187 were completely resistant to RSV infection but only to the respective virus . The protective activities of the peptides 174-187 were dependent on the Cys186-->Ser substitution . In addition, a recombinant protein representing the region 125-203 of the A subgroup G glycoprotein expressed in Escherichia coli was capable without further treatment to completely protect animals against RSV subgroup A infection . We show that the combinations of cysteinyl residues (positions 173, 176, 182, and 186) retained within either synthetic peptides or the recombinant protein G125-203 greatly influenced their protective activities . This indicates that the region 171-187 is essential for the protection conferred by the G125-203 protein . Furthermore, our results strongly suggest that the peptides' and recombinant protein's potencies are a function of a loop-like structure which is stabilized by intramolecular disulfide linkages between Cys176-Cys182 and Cys173-Cys186 . This is further supported by the observation that chemical blocking of the sulfidryl groups in synthetic peptides completely eliminated their protective activity. Vaccine, 1997 Mar, 15(4), 370 - 6 Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection; Weltzin R et al.; Intranasal (i.n.) delivery of antigen can be highly effective for generating circulating and secretory antibody responses . Mice were immunized i.n . with two antigens, human IgA, and Helicobacter pylori urease in the presence or absence of mucosal adjuvant . To restrict antigen delivery to the upper airways, protein solutions were administered in a small volume without anesthesia . Repeated daily i.n . administration of antigen without adjuvant elicited high levels of specific IgG in serum and IgA in serum, saliva, and feces . Once weekly i.n . immunization with co-administration of cholera toxin or Escherichia coli heat-labile toxin as adjuvant elicited somewhat lower levels of antibody to urease . When challenged with Helicobacter felis, only mice immunized with urease in the presence of adjuvant were protected against gastric infection. Jpn J Cancer Res, 1997 Mar, 88(3), 296 - 305 Efficient retrovirus-mediated cytokine-gene transduction of primary-cultured human glioma cells for tumor vaccination therapy; Wakimoto H et al.; In order to realize a novel vaccination treatment for malignant gliomas using tumor cells genetically modified to express certain cytokines, it is essential to achieve an efficient gene transduction into primary cultured cells . We investigated the feasibility of preparing a glioma vaccine through retrovirus-mediated gene transduction . Glioma cells were cultured primarily from surgically resected tumor tissues of six patients . We obtained more than 1000-fold proliferation of cultures within eight weeks in all six cases . In vitro infection with a recombinant retrovirus GKlacZ carrying an Escherichia coli beta-galactosidase marker gene resulted in over 65% gene transfer to the primary cultured glioma cells . Further enrichment (approximately 90%) of transduced cells was possible by employing repeated infections or using vectors with neo' marker gene . Two cytokine genes, granulocyte-macrophage colony-stimulating factor and interleukin-4, were introduced into glioma cells by sequential transduction with two single-expression GK vectors . The transduced glioma cells produced high levels of both cytokines . We also evaluated simultaneous introduction of two genes with double-expression GK vectors containing internal ribosomal entry site (IRES) or internal promoter (PGK) . Although the cells transduced with double-expression vectors secreted both cytokines, the level of the gene product following IRES or PGK was 10 times lower than that of the upstream gene product . The transduced cells retained cytokine secretion in vitro for 14 days after 100 Gy irradiation . Our data indicate the feasibility of retrovirus-mediated preparation of gene-modified tumor vaccines from clinical glioma materials, which could be useful for potentiating antitumor immunity in glioma patients. J Med Virol, 1997 Mar, 51(3), 159 - 66 Mutated epitopes of hepatitis B surface antigen fused to the core antigen of the virus induce antibodies that react with the native surface antigen; Shiau AL et al.; Fusion of peptide epitopes to the core antigen (HBcAg) of hepatitis B virus (HBV) enhances their immunogenicity, both quantitatively and qualitatively . In a number of vaccine-induced mutants of HBV, glycine145 of the surface antigen S polypeptide (HBsAg) has been replaced by arginine, resulting in loss of cross-reactivity with antibodies to normal (wild-type) HBsAg . HBcAg fusion proteins carrying the immunodominant epitope of HBsAg, in which glycine145 was replaced by arginine, glutamic acid, or lysine, were produced in Escherichia coli and formed particles that displayed HBc antigenicity and immunogenicity similar to that of HBcAg itself . The fusion proteins also elicited T-cell proliferative responsiveness to HBcAg and HBsAg . Fusions carrying either wild-type or mutated epitopes of HBsAG showed HBs antigenicity in immunoblot analysis and antigen-capture immunoradiometric assay, but both mutant and wild-type derivatives induced antibodies that cross-reacted with wild-type HBsAG . The results emphasise the potential for HBcAg fusion proteins in vaccines by broadening the antibody response in a way that could confer protection against both wild-type and variant form of HBV. Biophys J, 1997 Mar, 72(3), 1247 - 57 Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease; Lehtonen JY et al.; The well-characterized integral membrane protein lactose (lac) permease from Escherichia coli was reconstituted together with trace amounts (molar fraction X = 0.005 of the total phospholipid) of different pyrene-labeled phospholipid analogs into 1-palmitoyl-2-oleoyl-sn-glycero-3-sn-glycero-3-phospho-rac'-glycerol (POPG) liposomes . Effects of lac permease on bilayer lipid dynamics were investigated by measuring the excimer-to-monomer fluorescence intensity ratio IE/IM . Compared to control vesicles, the presence of lac permease (at a protein:phospholipid stoichiometry P/L of 1:4.000) increased the rate of excimer formation by 1-palmitoyl-2{6-(pyren-1-yl)}decanoyl-sn-glycero-3-phosphocholine (PPDPC) by approximately fivefold . Decreasing P/L from approximately 1:4.000 to 1:7.600 decreased the IE/IM for PPDPC from 0.16 to 0.05, respectively . An increase in bilayer fluidity due to permease is unlikely, thus implying that the augmented IE/IM should arise from partial lateral segregation of PPDPC in the vesicles . This notion is supported by the further 38% increase in IE/IM observed for the pyrene-labeled Cys-148 lac permease reconstituted into POPG vesicles at P/L 1:4000 . The importance of the length of the lipid-protein boundary is implicated by the reduction in IE/IM resulting from the aggregation of the lac permease in vesicles by a monoclonal antibody . Interestingly, excimer formation by 1-palmitoyl-2{6-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) was enhanced only fourfold in the presence of lac permease . Results obtained with the corresponding pyrenyl phosphatidylglycerols and -methanols were qualitatively similar to those above, thus indicating that lipid headgroup-protein interactions are not involved . Inclusion of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N-(5-fluoresce inthio- carbamoyl) (DPPF, X = 0.005) into reconstituted lactose permease vesicles containing PPDPC caused a nearly 90% decrease in excimer fluorescence, whereas in control vesicles lacking the reconstituted protein only 40% quenching was evident . The addition of 1,2-dipalmitoyl-sn-glycero-3-phospho-rac'-glycerol (DPPG) decreased IE/IM for PPDPC, revealing the driving force for the lateral segregation of this probe to become attenuated . More specifically for protein-free bilayers at XDPPG = 0.10 the rate of lateral diffusion of PPDPC in POPG is diminished, as evidenced by the 24% decrement in IE/IM, under these conditions the increase in IE/IM due to lac permease was strongly reduced, by approximately 84% . The present data are interpreted in terms of the hydrophobic mismatch theory, which predicts that integral membrane proteins will draw lipids of similar hydrophobic thickness into their vicinity . In brief, the approximate lengths of most of the predicted 12 hydrophobic, membrane-spanning alpha-helical segments of lactose permease range between 28.5 and 37.5 A and thus exceed the hydrophobic thickness of POPG of approximately 25.8 A . Therefore, to reduce the free energy of the assembly, longer lipids such as PPDPC and DPPF are accumulated in the immediate vicinity of lactose permease in fluid, liquid crystalline POPG bilayers. Biophys J, 1997 Mar, 72(3), 1109 - 26 A novel method for structure-based prediction of ion channel conductance properties; Smart OS et al.; A rapid and easy-to-use method of predicting the conductance of an ion channel from its three-dimensional structure is presented . The method combines the pore dimensions of the channel as measured in the HOLE program with an Ohmic model of conductance . An empirically based correction factor is then applied . The method yielded good results for six experimental channel structures (none of which were included in the training set) with predictions accurate to within an average factor of 1.62 to the true values . The predictive r2 was equal to 0.90, which is indicative of a good predictive ability . The procedure is used to validate model structures of alamethicin and phospholamban . Two genuine predictions for the conductance of channels with known structure but without reported conductances are given . A modification of the procedure that calculates the expected results for the effect of the addition of nonelectrolyte polymers on conductance is set out . Results for a cholera toxin B-subunit crystal structure agree well with the measured values . The difficulty in interpreting such studies is discussed, with the conclusion that measurements on channels of known structure are required. Biophys J, 1997 Mar, 72(3), 1031 - 46 Molecular dynamics simulations of six different fully hydrated monomeric conformers of Escherichia coli re-lipopolysaccharide in the presence and absence of Ca2+; Obst S et al.; Six previously published conformational models of Escherichia coli Re lipopolysaccharide (ReLPS) were subjected to molecular dynamics simulations using the CHARMM force field . The monomers of ReLPS were completely immersed in a water box . The dynamic behavior of the solvated models in the presence and absence of calcium cations was compared . The structure of the solvent shell was analyzed in terms of radial distribution functions . Diffusion coefficients and mean residence times were analyzed to characterize the dynamic behavior of the solvent . Order parameters and number of gauche defects were used for the description of the dynamics of the acyl chains . The cations are preferentially located between the carboxylate and phosphate groups of the headgroup . Their presence leads to a rigidification of the headgroup structure and alters the conformation of the backbone, thus influencing the structure and flexibility of the hydrophobic region as well . The effect of calcium on the backbone flexibility was measured in terms of glycosidic torsion angles . The six fatty acid chains of each ReLPS monomer adopt a highly ordered micromembrane structure . The packing parameter indicates that aggregation of these ReLPS monomers will lead to lamellar structures . Evaluation of all data enables us to present one conformation, C, which is thought to best represent the average structure of the ReLPS conformers. J Biochem (Tokyo), 1997 Mar, 121(3), 456 - 63 The tissue-type plasminogen activator inhibitor ETIa from Erythrina variegata: structural basis for the inhibitory activity by cloning, expression, and mutagenesis of the cDNA encoding ETIa; Kouzuma Y et al.; Erythrina variegata trypsin inhibitor ETIa belongs to the Kunitz inhibitor family, but is unique in its ability to bind and inhibit tissue-type plasminogen activator (tPA) . A cDNA clone encoding ETIa was isolated from the lambda gt11 cDNA library using specific antiserum as a probe and characterized by nucleotide sequencing . The cloned ETIa cDNA consists of 762 nucleotides and includes an open reading frame encoding a polypeptide of 198 amino acids . Comparison of the deduced protein sequence and the determined protein sequence indicated the presence of two signal peptides composed of 24 and 2 amino acids at the N- and C-termini, respectively . The cDNA encoding mature ETIa was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b,