bluuebunny/crossref_metadata_2025_split huggingface dataset:
Unusual zymogen‐processing properties of a mutated form of prochymosin
Authors: McCaman Michael T. , Cummings Diana Begley 
Published: May 2005

AbstractSite‐specific mutagenesis of the gene encoding bovine prochymosin was used to produce a mutated zymogen in which seven contiguous amino acids of the N‐terminal propeptide had been deleted and an eighth residue had been substituted. This altered region spans the normal site of autocatalytic proteolysis that occurs at the same time as (enzymatic) activation of prochymosin at acidic pH. Activation of the mutated zymogen at pH 4.5 was extremely slow, and cleavage occurred at an unusual Ser‐Lys bond in the prochymosin incubated at pH 2 generated the usual pseudochymosin by cleavage of the normal Phe‐Leu bond, but at a rate severalfold slower than the authentic zymogen. These results indicate that even after deletion of seven of 42 amino acids of the propeptide the mutant protein could still assume a prochymosin (zymogen) structure, although these changes did result in striking differences in acid‐catalyzed activation and processing reactions at one but not the other of the two processing sites of prochymosin.
Conformation of alamethicin in phospholipid vesicles: Implications for insertion models
Authors: Cascio M. , Wallace B. A. 
Published: May 2005

AbstractThe secondary structure of alamethicin, a membrane channel‐forming polypeptide, has been examined by circular dichroism spectroscopy to determine the relationship of its conformation in organic solution to its conformation in a membrane‐bound state. The spectrum of alamethicin in small unilamellar dimyristoyl phosphatidylcholine vesicles is significantly different from its spectrum in 10% methanol/acetonitrile, the solvent from which it was crystallized (Fox and Richards: Nature 300:325–330, 1982), as well as its spectrum in methanol, the solvent in which NMR studies have been done (Banerjee and Chan: Biochemistry 22:3709–3713, 1983). This suggests that structural models based on studies of the molecule in organic solvents may not be entirely appropriate for the membrane‐bound state. To distinguish between different models for channel formation and insertion, two different methods were used to associate the alamethicin with vesicles; in addition, the effect of oligomerization on the conformation of the membrane‐bound state was investigated. These studies are consistent with a modified insertion model in which alamethicin monomers, dimers, or trimers associate with the bilayer and then spontaneously oligomerize to form a prechannel with a higher helix content. This aggregate could then “open” upon application of an appropriate gating transmembrane potential.
Internal motion of a tryptophan residue in Streptomyces subtilisin inhibitor: Deuterium nuclear magnetic resonance in solution
Authors: Akasaka Kazuyuki , Inoue Tomoko , Tamura Atsuo , Watari Hiroshi , Abe Kimie , Kainosho Masatsune 
Published: May 2005

AbstractDeuterium NMR spectroscopy was used to study internal motions of a deuterium‐labeled single tryptophan (Trp) residue (per subunit) of Streptomyces subtilisin inhibitor (SSI) in solution. The free inhibitor with the five ring protons of the Trp replaced with deuterons showed a narrow resonance component (56 Hz) of about one‐quarter of the total intensity, in addition to the broad resonance component (about 600 Hz) at 25°C, showing that it exits in an equilibrium mixture of two conformers, in one of which the typtophan side chain is highly mobile. In analogy to the two structures of SSI found in the crystal, these two conformers were attributed to the one in which the contact between the α‐lobe and the beta;‐lobe of the subunit is tight and the other in which the same contact is loose. When SSI forms a complex with subtilisin BPN′, the broad component becomes invisibly broad, but the narrow component increases with even further narrowing, suggesting that the binding to the enzyme favors the “loose” conformer over the “tight” conformer.
Structural alignment and analysis of two distantly related proteins: Aplysia limacina myoglobin and sea lamprey globin
Authors: Pastore Annalisa , Lesk Arthur M. , Bolognesi Martino , Onesti Silvia 
Published: May 2005

AbstractTwo new globin structures have recently been determined at high resolution: the globin from the mollusc Aplysia limacina at 1.6 A resolution and a new refinement of the structure from sea lamprey. Two amino acid sequences of these homologous molecules have only 30% residue identity in an optimal alignment. We discuss some of the problems arising in the alignment of Aplysia globin with other globins of known structure, a challenging problem because of the distant relationship. Four independent approaches were applied to the alignment of the Aplysia and lamprey globins, including those based on individual sequence comparisons, structural analysis, and the relatively new method of templates or fingerprints derived for an entire family of proteins. We also compare these two new structures with what is already known about the globin family. A detailed description of the two structures shows that the two molecules contain the main structural features common to all the globins so far studied with several minor but interesting hitherto unobserved variations.
Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: A pentacoordinated binding of the SCN− ion to the zinc at high pH
Authors: Eriksson A. Elisabeth , Kylsten Per M. , Jones T. Alwyn , Liljas Anders 
Published: May 2005

AbstractThe binding of four inhibitors—mercuric ion, 3‐acetoxymercuri‐4‐aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion—to human carbonic anhydrase II (HCA II) has been studied with X‐ray crystallography.The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 Å resolution. Mercuric ions are observed at both nitrogens in the His‐64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys‐206.The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 Å, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closet to the zinc ion is 3.1 Å away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc.The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 Å resolution using the least‐squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN− ion is 1.9 Å from the zinc ion but shifted 1.3 Å with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the Oγl atom of Thr‐199. This is due to the inability of the Oγl atom of Thr‐199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN− molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so‐called “deep” water molecule of the native enzyme. The zinc‐bound water molecule is 2.2 Å from the zinc ion and 2.4 Å from the SCN− nitrogen. In addition, this water is hydrogen bonded to the Oγl atom of Thr‐199 and to another water molecule.We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3− ion are ligated to the zinc ion.
Comparison of CD spectra in the aromatic region on a series on variant proteins substituted at a unique position of tryptophan synthase α‐subunit
Authors: Ogasahara Kyoko , Sawada Shintaro , Yutani Katsuhide 
Published: May 2005

AbstractCD spectra in the aromatic region of a series of the mutant α‐subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25°C. The measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild‐type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild‐type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of thewild‐type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild‐type proteins. These residues are polar. In the third group, the CDvalues of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild‐type protein, except for oneband at 276.5 nm. these results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residuesat position 49.
The 15 N‐terminal amino acids of hexokinase II are not required for in vivo function: Analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae
Authors: Ma Hong , Bloom Leslie M. , Dakin Susan E. , Walsh Christopher T. , Botstein David 
Published: May 2005

AbstractThe function of the N‐terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form).This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short from protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild‐type protein dimerizes. Kinetic studies show that the enzymatic activities are very similarto wild‐type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form ofthe enzyme is similar to the wild‐type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N‐terminalamino acids of hexokinase II are not required in vivo either for phosporylation of hexoses or for glucose repression.
Substrate specificities in class A β‐lactamases: Preference for penams vs. cephams. The role of residues 237
Authors: Healey William J. , Labgold Marc R. , Richards John H. 
Published: May 2005

AbstractSite saturation mutagenesis has been carried out at Ala‐237 in RTEM‐1 β‐lactamase to assess the role of this site in modulating differences in specificity of β‐lactamases for penams vs. cephams as substrates. (An Ala‐237 Thr mutation had previously been shown to increase activity on cephems by about 30–80%.1,2) Screening of all 19 possibles mutants on penams and cephems revealed the even more active Ala‐237 Asn mutant. Detailed kinnetic analysis showns that this mutant has about four times the activity toward cephalothin and cephalosporin C as the wild‐type enzyme. Both mutations reduce the activity toward penams to about 10% that of RETM‐1 β‐lactamase and lower by about 5°C the tempreature at which the enzyme denatures. Functional properties of the other mutants have also been surveyed. The most intresting aspect of these results is that two quite disparate amino acids, theronine and asparagine, when intorduced for Ala‐237, cause such similar changes in enzyme specificity while more similar residues do not alter the catalytic properties of the enzyme to such a significant degree.
Deletions and replacements of omega loops in yeast iso‐1‐cytochrome c
Authors: Fetrow Jacquelyn S. , Cardillo Thomas S. , Sherman Fred 
Published: May 2005

AbstractΩ(Omega)‐loops are protein secondary structural elements having small distance between segment termini. It should be possible to delete or replace certain of these Ω‐loops without greatly distorting the overall structure of the remaining portion of the molecule. Functional requirements of regions of iso‐1‐cytochrome c from the yeast Saccharomyces cerevisiae were in investigated by determining the biosynthesis and activity in vivo of mutant forms in which four different Ω‐loops were individually deleted, or in which one Ω‐loop was replaced with five different segments. Deletion encompassing amino acid positions 27–33 and79–83 either prevented synthesis of the holoprotein, or produced highly labile iso‐1‐cytochromes c, whereas deletions encompassing position 42–45 and 48–55 allowed partial synthesis and activity. These two latter regions, therefore, are not absolutely required for any biosynthetic process such as heme attachment, mitochondrial import, or for enzymatic interactions. All replacements in Loop A (residue position 24–33) with the same size (10 amino acid residues), longer (13 and 15 amino acid residues), or shorter segments (6 amino acid residues), resulted in strains having at least partial levels of iso‐1‐cytochrome c; however, the relative activities ranged from zero to almost the normal level. Thus, Loop A does not appear to be essential for such biosynthetic steps as heme attachment and mitochondrial import. In contrast, the full range of relative activities suggest that this region interacts with physiological partners to carry out efficient electron transport.
Structural determinants of the conformations of medium‐sized loops in proteins
Authors: Tramontano Anna , Chothia Cyrus , Lesk Arthur M. 
Published: May 2005

AbstractLoops are integral components of protein structures, providing links between elements of secondary structure, and in many cases contributing to catalytic and binding sites.The conformations of short loops are now understood to depend primarily on their amino acid sequences. In contrast, the structural determinants of longer loops involve hydrogen‐bonding and packing interactions within the loop and with other parts of the protein. By searching solved protein structures for regions similar in main chain conformation to the antigen‐binding loops in immunoglobulins, we identified medium‐sized loops of similar structure in unrelated proteins, and compared the determinants of their conformations.For loops that form compact substructures the major determinant of the conformation is the formation of hydrogen bonds to inward‐pointing main chain atoms. For oops that have more extended conformations, the major determinant of their structure is the packing of a particular residue or residues against the rest of the protein.The following picture emerges: Medium‐sized lops of similar conformation are stabilized by similar interaction. The groups that interact with the loop have very similar spatial dispositions with respect to the loop. However, the residues that provide these interactions may arise from dissimilar parts of the protein: The conformation of the loop requires certain interactions that the protein may provide in a variety of ways.
A mutant T4 lysozyme (Val 131 → Ala) designed to increase thermostability by the reduction of strain within an α‐helix
Authors: Dao‐Pin S. , Baase W. A. , Matthews B. W. 
Published: May 2005

AbstractAn attempt has been made to identify residues in T4 phage lyoszyme that may have strained conformations and, by appropriate site‐directed replacements, to reduce this strain and thus increase the thermostability of the protein. Valine 131, within α‐helix 126–134, was identified as a potential candidate. Its side‐chain rotational as a potential candidate. Its side‐chain rotation angle, χ1, differs by approximately 18° from the low‐energy trans configuration. In addition, it is largely solvent exposed, yet is held in a rigid conformation. The mutant protein with Val 131 replaced by alanine temperature 0.9°C higher than that of wildtype lyoszyme at pH 2.8. As a control, The mutant Val 131 → Thr was also constructed and its melting temperature was found to be marginally lower than wild type. Higher‐resolution crystal structure determination of the mutant lysozymes show that their structure are virtually identical with that of wild‐type lyoszyme, except for the Val → Ala or Val → Thr replacement. Analysis of the different structures suggests that the design of the Val→Ala substitution was, in principle, successful, although the apparent gain in stability caused by reduction in strain is modest and is somewhat offset by the loss of hydrophobic interactions and by entrophic effects. The results also help to provide a structural retionalization that alanine has a higher helix propensity than valine or theronine.
Identification of protein folds: Matching hydrophobicity patterns of sequence sets with solvent accessibility patterns of known structures
Authors: Bowie James U. , Clarke Neil D. , Pabo Carl O. , Sauer Robert T. 
Published: May 2005

AbstractHydrophobic side chains often are buried in the interior of a protein, and evolutionarily related proteins usually maintain the hydrophobic character of buried position. In this paper we shown that a pattern of hydrophobicity values derived from a set of related protein sequences is well correlated with the linear pattern of side‐chain solvent accessibility values, calculated from a known protein structure representative of the sequences. In several cases, information from aligned sequences can be used to select the correct tertiary fold from a large database of protein structures.
HERA—A program to draw schematic diagrams of protein secondary structures
Authors: Hutchinson E. Gail , Thornton Janet M. 
Published: May 2005

AbstractA program is described which generates hydrogen bonding diagrams of protein structures and optionally helical wheels and helical nets. The program can also be used simply to calculate the connectivities of β‐strands and to automatically extract simple structural motifs such as hairpins or Greek keys. The program greatly reduces the effort required to produce these diagrams and offers considerable flexibility in the information which can be represented. The usefulness of the program is illustrated by several examples including comparing homologous families, correlating protein structure with attributes of individual residues, and extracting all examples of the ψ‐loop motif from the Brookhaven Data Bank.
Three‐Dimensionnal structures of complexes of Lathyrus ochrus isolectin I with glucose and mannose: Fine specificity of the monosaccharide‐binding site
Authors: Bourne Yves , Roussel Alain , Frey Michel , Rougé Pierre , Fontecilla‐Camps Juan‐C , Cambillau Christian 
Published: May 2005

AbstractThe structure of the methyl‐α‐D‐mannopyranoside–LOL I complex has been solved by the molecular replacement method using the refined saccharide‐free LOL I coordinates as starting model. The methyl‐α‐D‐mannopyranoside–LOL I complex was refined by simulated annealing using the program X‐PLOR. The final R‐factor value is 0.182 [Fo > 1σ(Fo)]. The isostructural methyl‐α‐D‐glucopyranoside‐LOL I complex was refined by X‐Ray coupled energy minimization using the methyl‐α‐D‐mannopyranoside‐LOL I structure as a starting model to an R factor of 0.179 (all data). In both crystal forms, each dimer binds two molecules of sugar in pockets found near the calcium ions. The two saccharide moieties, which are in the C1 chair conformation, establish the same hydrogen bond pattern with the lectin. However, the van der Walls contacts are different between the O2, C2, C6, and O6 atoms of the two molecules and the backbone atoms of residues 208–211. Mannose, due to its axial C2 conformation, encloses the backbone atoms of the protein in a clamplike way. Van der Waals energy calculations suggest that this better complementarity of the mannoside molecule with the lectin could explain its higher affinity for isolectin I.
Design and synthesis of the pseudo‐EF hand in calbindin D9K: Effect of amino acid substitutions in the α‐helical regions
Authors: Tsuji Takashi , Kaiser Emil T. 
Published: May 2005

AbstractA series of 37‐residue analogues of the pseudo‐EF hand in bovine calbindin D9K has been synthesized by the solid phase method. In the presence of calcium an α‐helical induction of up to 44% was observed for the peptide with the native sequence with a Kd for calcium binding of 0.35 mM. A number of amino acid substitutions have been carried out to study the packing of the two α‐helices based on the crystal structure of the entire protein. Three strategies were employed: (1) replacement of the Leu residues, which in the crystal structure do not contribute to the hydrophobic interaction between the two helices, by Gln or Ala in order to control the orientation of the helix packing, (2) stabilization of the individual helix by introducing a Glu−…︁Lys+ salt bridge or by changing the N‐terminal charge to compensate for the helix dipole moment, and (3) introduction of a disulfide bond between the two helices to help the packing of the helices.The mutants with the substitution of (Leu‐30, Leu‐32) to (Gln‐30, Gln‐32), (Gln‐30, Ala‐32), and (Ala‐30, Ala‐32) designed based on the strategy 1 do not show any affinity for calcium and have low α‐helicity. The Leu‐30 to Lys‐30 mutant designed to form a salt bridge between the side chains of Glu‐26 and Lys‐30 has an apparent Kd for calcium of 6.8 mM. Kd of the N‐terminal acetylated and succinylated mutants are 0.41 and 0.45 mM, respectively, and no increase in the α‐helix content relative to that of the natural sequence peptide is observed. The disulfide containing mutants, namely Tyr‐13, Leu‐31 to Cys‐31 and Tyr‐13, Leu‐31 to Cys‐13, hCys‐31, show apparent Kd values of 0.93 and 2.1 mM, respectively. The former mutant shows the highest α‐helix content among the peptides studied in the presence and absence of calcium. While it is difficult to construct an isolated and rigid helix–loop–helix motif with peptides of this size, introduction of a disulfide bond proved to be effective for this purpose.