bluuebunny/crossref_metadata_2025_split huggingface dataset:
Response of dynamic structure to removal of a disulfide bond: Normal mode refinement of C77A/C95A mutant of human lysozyme
Authors: Kidera Akinori , Inaka Koji , Matsushima Masaaki , Gō Nobuhiro 
Published: July 2010

AbstractIn order to investigate the response of dynamic structure to removal of a disulfide bond, the dynamic structure of human lysozyme has been compared to its C77A/C95A mutant. The dynamic structures of the wild type and mutant are determined by normal mode refinement of 1.5‐Å‐resoltion X‐ray data. The C77AK95A mutant shows an increase in apparent fluctuations at most residues. However, most of the change originates from an increase in the external fluctuations, reflecting the effect of the mutation on the quality of crystals. The effects of disulfide bond removal on the internal fluctuations are almost exclusively limited to the mutation site at residue 77. No significant change in the correlation of the internal fluctuations is found in either the overall or local dynamics. This indicates that the disulfide bond does not have any substantial role to play in the dynamic structure. A comparison of the wild‐type and mutant coordinates suggests that the disulfide bond does not prevent the 2 domains from parting from each other. Instead, the structural changes are characteristic of a cavity‐creating mutation, where atoms surrounding the mutation site move cooperatively toward the space created by the smaller alanine side chain. Although this produces tighter packing, more than half of the cavity volume remains unoccupied, thus destabilizing the native state.
Crystal structure of cholera toxin B‐pentamer bound to receptor GM1 pentasaccharide
Authors: Merritt Ethan A. , Sarfaty Steve , Akker Focco Van Den , L'Hoir Cécile , Martial Joseph A. , Hol Wim G.J. 
Published: July 2010

AbstractCholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B‐pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B‐pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B‐subunit, with a single solvent‐mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N‐acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2‐fingered grip: the Gal(β1‐3)CalNAc moiety representing the “forefinger” and the sialic acid representing the “thumb.” The residues forming the binding site are conserved between cholera toxin and the homologous heat‐labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than Gm1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1‐mediated signal transduction.
Role of the Cys 2‐Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1
Authors: Mayr Lorenz M. , Willbold Dieter , Landt Olfert , Schmidm Franz X. 
Published: July 2010

AbstractThe Cys 2‐Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease Fl. This C2S/ClON variant resembles the wild‐type protein in structure and in catalytic activity. Minor structural changes were observed by 2‐dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2‐Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and thetranstocisisomerizations of Pro 39 and Pro 55 are still the rate‐limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild‐type protein and the C2S/ClON variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2‐Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild‐type protein.
Real‐time DNA binding measurements of the ETSl recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms
Authors: Fisher Robert J. , Fivash Matthew , Casas‐Finet Jose , Erickson John W. , Kondoh Akiko , Bladen Sharon V. , Fisher Constance , Watson Dennis K. , Papas Takis 
Published: July 2010

AbstractThe sequence‐specific DNA binding of recombinant p42 and p51 ETSl oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETSl or the phosphorylation of expressed Exon VII in p51 ETSl had an effect on DNA binding activity. The kinetics of sequence‐specific DNA binding was measured using real‐time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real‐time binding of p42 and p51 ETSl displayed significant differences in kinetic behavior. p51 ETSl is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETSl exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETSl DNA binding states are characterized by rapid turnover, whereas the p42 ETSl DNA binding states are 4‐20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETSl with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETSl as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETSl by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETSl sequence‐specific DNA binding occurs through phosphorylation by a calcium‐dependent second messenger. The p42 ETSl lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.
An 1H NMR determination of the three‐dimensional structures of mirror‐image forms of a Leu‐5 variant of the trypsin inhibitor from Ecballium elaterium (EETI‐II)
Authors: Nielsen Katherine J. , Alewood Dianne , Andrews John , Kent Stephen B.H. , Craik David J. 
Published: July 2010

AbstractThe 3‐dimensional structures of mirror‐image forms of a Leu‐5 variant of the trypsin inhibitor Ecballium elaterium (EETI‐II) have been determined by 1H NMR spectroscopy and simulated annealing calculations incorporating NOE‐derived distance constraints. Spectra were assigned using 2‐dimensional NMR methods at 400 MHz, and internuclear distances were determined from NOESY experiments. Three‐bond spin‐spin couplings between CαH and amide protons, amide exchange rates, and the temperature dependence of amide chemical shifts were also measured. The structure consists largely of loops and turns, with a short region of β‐sheet. The Leu‐5 substitution produces a substantial reduction in affinity for trypsin relative to native EETI‐II, which contains an Ile at this position. The global structure of the Leu‐5 analogue studied here is similar to that reported for native EETI‐II (Heitz A, Chiche L, Le‐Nguyen D, Castro B, 1989, Biochemistry 28:2392‐2398) and to X‐ray and NMR structures of the related proteinase inhibitor CMTI‐I (Bode W et al., 1989, FEBS Lett 242:285‐292; Holak TA et al., 1989a, J Mol Biol 210:649‐654; Holak TA, Condol D, Otlewski J, Wilusz T, 1989b, J Mol Biol 210:635‐648; Holak TA, Habazettl J, Oschkinat H, Otlewski J, 1991, J Am Chem Soc 113:3196‐3198). The region near the scissile bond is the most disordered part of the structure, based on geometric superimposition of 40 calculated structures. This disorder most likely reflects additional motion being present in this region relative to the rest of the protein. This motional disorder is increased in the Leu‐5 analogue relative to the native form and may be responsible for its reduced trypsin binding. A second form of the protein synthesized with all (d) amino acids was also studied by NMR and found to have a spectrum identical with that of the (l) form. This is consistent with the (d) form being a mirror image of the (l) form and not distinguishable by NMR in an achiral solvent (i.e., H2O). The (d) form has no activity against trypsin, as would be expected for a mirror‐image form.
Acid sphingomyelinase possesses a domain homologous to its activator proteins: Saposins B and D
Authors: Ponting Christopher P. 
Published: July 2010

AbstractAn N‐terminal region of the acid sphingomyelinase sequence (residues 89‐165) is shown to be homologous to saposintype sequences. By analogy with the known functions of saposins, this sphingomyelinase saposin‐type domain may possess lipid‐binding and/or sphingomyelinase‐activator properties. This finding may prove to be important in the understanding of Niemann‐Pick disease, which results from sphingomyelinase deficiency.
Autonomous subdomains in protein folding
Authors: Wu Lawren C. , Grandori Rita , Carey Jannette 
Published: July 2010

AbstractProteolytic dissection of native trp repressor and horse heart cytochrome c has been used to infer some of the steps in the folding pathways of the intact proteins. For both proteins, small fragments are capable of undergoing spontaneous noncovalent association to form subdomains with native‐like secondary and/or tertiary structural features, suggesting that dissection/reassembly may be a general method to gain insight into the structures of folding intermediates. The importance of this approach is its simplicity and potential applicability to studying the folding pathways of a wide range of proteins. The proteases report on the structure and dynamics of the native state, circumventing the need for prior knowledge of the structures of folding intermediates. The observation that small fragments of proteins can associate noncovalently suggests that protein folding can be viewed as an intramolecular “recognition” process. The results imply that substantial information about protein structure and folding is encoded at the level of subdomains, and that chain connectivity has only a minor role in determining the fold.
Residues 36‐42 of liver RNase PL3 contribute to its uridine‐preferring substrate specificity. Cloning of the cDNA and site‐directed mutagenesis studies
Authors: Vicentini Anna M. , Hemmings Brian A. , Hofsteenge Jan 
Published: July 2010

AbstractWithin the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase. Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture. The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N‐terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3′,5′)adenosine and cytidylyl(3′,5′)adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5′ side of the scissile bond. Replacement, by site‐directed mutagenesis, of residues 36‐42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine‐containing substrates. This suggests that this region of the molecule contains some of the elements that determine substrate specificity.
Modular arrangement of proteins as inferred from analysis of homology
Authors: Sonnhammer Erik L.L. , Kahn Daniel 
Published: July 2010

AbstractThe structure of many proteins consists of a combination of discrete modules that have been shuffled during evolution. Such modules can frequently be recognized from the analysis of homology. Here we present a systematic analysis of the modular organization of all sequenced proteins. To achieve this we have developed an automatic method to identify protein domains from sequence comparisons. Homologous domains can then be clustered into consistent families. The method was applied to all 21,098 nonfragment protein sequences in SWISS‐PROT 21.0, which was automatically reorganized into a comprehensive protein domain database, ProDom. We have constructed multiple sequence alignments for each domain family in ProDom, from which consensus sequences were generated. These nonredundant domain consensuses are useful for fast homology searches. Domain organization in ProDom is exemplified for proteins of the phosphoenolpyruvate: sugar phosphotransferase system (PEP:PTS) and for bacterial 2‐component regulators. We provide 2 examples of previously unrecognized domain arrangements discovered with the help of, ProDom.
The importance of anchorage in determining a strained protein loop conformation
Authors: Hodel Alec , Kautz Roger A. , Adelman David M. , Fox Robert O. 
Published: July 2010

AbstractWe examine the role of the conformational restriction imposed by constrained ends of a protein loop on the determination of a strained loop conformation. The Lys 116‐Pro 117 peptide bond of staphylococcal nuclease A exists in equilibrium between the cis and trans isomers. The folded protein favors the strained cis isomer with an occupancy of 90%. This peptide bond is contained in a solvent‐exposed, flexible loop of residues 112‐117 whose ends are anchored by Val 111 and Asn 118. Asn 118 is constrained by 2 side‐chain hydrogen bonds. We investigate the importance of this constraint by replacing Asn 118 with aspartate, alanine, and glycine. We found that removing 1 or more of the hydrogen bonds observed in Asn 118 stabilizes the trans configuration over the cis configuration. By protonating the Asp 118 side chain of N118D through decreased pH, the hydrogen bonding character of Asp 118 approached that of Asn 118 in nuclease A, and the cis configuration was stabilized relative to the trans configuration. These data suggest that the rigid anchoring of the loop end is important in establishing the strained cis conformation. The segment of residues 112‐117 in nuclease A provides a promising model system for study of the basic principles that determine polypeptide conformations. Such studies could be useful in the rational design or redesign of protein molecules.
Analysis of protein transmembrane helical regions by a neural network
Authors: Dombi George W. , Lawrence Jeannette 
Published: July 2010

AbstractNeural networks were used to generalize common themes found in transmembrane‐spanning protein helices. Various‐sized databases were used containing nonoverlapping sequences, each 25 amino acids long. Training consisted of sorting these sequences into 1 of 2 groups: transmembrane helical peptides or nontransmembrane peptides. Learning was measured using a test set 10% the size of the training set. As training set size increased from 214 sequences to 1,751 sequences, learning increased in a nonlinear manner from 75% to a high of 98%, then declined to a low of 87%. The final training database consisted of roughly equal numbers of transmembrane (928) and nontransmembrane (1,018) sequences. All transmembrane sequences were entered into the database with respect to their lipid membrane orientation: from inside the membrane to outside. Generalized transmembrane helix and nontransmembrane peptides were constructed from the maximally weighted connecting strengths of fully trained networks. Four generalized transmembrane helices were found to contain 9 consensus residues: a K‐R‐F triplet was found at the inside lipid interface, 2 isoleucine and 2 other phenylalanine residues were present in the helical body, and 2 tryptophan residues were found near the outside lipid interface. As a test of the training method, bacteriorhodopsin was examined to determine the position of its 7 transmembrane helices.
Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen
Authors: Fisher Cindy L. , Greengard Judith S. , Griffin John H. 
Published: July 2010

AbstractThree‐dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site‐directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3‐dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.
Heparin binding domain peptides of antithrombin III: Analysis by isothermal titration calorimetry and circular dichroism spectroscopy
Authors: Tyler‐Cross Ruth , Harris Robert B. , Sobel Michael , Marques Dalila 
Published: July 2010

AbstractThe serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F123‐G148, which encompasses both the purported high‐affinity pentasaccharide binding region and an adjacent, C‐terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino‐terminal truncations of this sequence, including L130‐G148 and K136‐G148 displayed attenuated heparin binding activities. In fact, K136‐G148 appears to encompass only a low‐affinity heparin binding site. In contrast, peptides based solely on the high‐affinity binding site (K121‐A134) displayed much higher affinities for heparin. By CD spectrometry, these high‐affinity peptides are chiefly random coil in nature, but low μM concentrations of heparin induce significant α‐helix conformation. K121‐A134 also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure‐function relations of heparin‐ATIII interactions.
The three‐dimensional profile method using residue preference as a continuous function of residue environment
Authors: Zhang Kam Y.J. , Eisenberg David 
Published: July 2010

AbstractIn the 3‐dimensional profile method, the compatibility of an amino acid sequence for a given protein structure is scored as the sum of the preferences of the residues for their environments in the 3D structure. In the original method (Bowie JU, Lüthy R, Eisenberg D, 1991, Science 253:164‐170), residue environments were quantized into 18 discrete environmental classes. Here, amino acid residue preferences are expressed as a continuous function of environmental variables (residue area buried and fractional area buried by polar atoms). This continuous representation of residue preferences, expressed as a Fourier series, avoids the abrupt change of preference of residues in slightly different environments, as encountered in the original method with its 18 discrete environmental classes. When compared with the discrete 18‐class representation of residue environments, this continuous 3D profile is found to be more sensitive in identifying sequences that fold into the profiled structure but share with it little sequence identity. The continuous 3D profile is also less sensitive to errors in environmental variables than is the discrete 3D profile. The continuous 3D profile can also be used to detect wrong folds or incorrectly modeled segments in an otherwise correct structure, as could the discrete 3D profile (Lüthy R, Bowie JU, Eisenberg D, 1992, Nature 356:83‐85). Moreover, the progress of structure improvement during atomic refinement can also be monitored by examining the profile scores in a moving‐window scan. Finally, by defining a functional form for profile scores, we open the way to profile atomic refinement in which an atomic structure adjusts to produce residue environments more compatible with the protein side chains.
Similarity between pyridoxal/pyridoxamine phosphate‐dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes
Authors: Pascarella Stefano , Bossa Francesco 
Published: July 2010

AbstractA multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate‐dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate‐dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.