bluuebunny/crossref_metadata_2025_split huggingface dataset:
Folding and aggregation of TEM β‐lactamase: Analogies with the formation of inclusion bodies in Escherichia coli
Authors: Georgiou George , Valax Pascal , Ostermeier Marc , Horowitz Paul M. 
Published: February 2009

AbstractThe enzyme TEM β‐lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM β‐lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0–1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis‐1‐anilino‐8‐naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM β‐lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0–1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM β‐lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM β‐lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131–136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.
Quantification of the calcium‐induced secondary structural changes in the regulatory domain of troponin‐C
Authors: Gagné Stéphane M. , Tsuda Sakae , Li Monica X. , Chandra Murali , Smillie Lawrence B. , Sykes Brian D. 
Published: February 2009

AbstractThe backbone resonance assignments have been completed for the apo (1H and 15N) and calcium‐loaded (1H, 15N, and 13C) regulatory N‐domain of chicken skeletal troponin‐C (1–90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical‐shift information, along with detailed NOE analysis and 3JHNHα coupling constants, permitted the determination and quantification of the Ca2+‐induced secondary structural change in the N‐domain of TnC. For both structures, 5 helices and 2 short β‐strands were found, as was observed in the apo N‐domain of the crystal structure of whole TnC (Herzberg O, James MNG, 1988, J Mol Biol 203:761–779). The NMR solution structure of the apo form is indistinguishable from the crystal structure, whereas some structural differences are evident when comparing the 2Ca2+ state solution structure with the apo one. The major conformational change observed is the straightening of helix‐B upon Ca2+ binding. The possible importance and role of this conformational change is explored. Previous CD studies on the regulatory domain of TnC showed a significant Ca2+‐induced increase in negative ellipticity, suggesting a significant increase in helical content upon Ca2+ binding. The present study shows that there is virtually no change in α‐helical content associated with the transition from apo to the 2Ca2+ state of the N‐domain of TnC. Therefore, the Ca2+‐induced increase in ellipticity observed by CD does not relate to a change in helical content, but more likely to changes in spatial orientation of helices.
Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions
Authors: Monera Oscar D. , Kay Cyril M. , Hodges Robert S. 
Published: February 2009

AbstractThe objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled‐coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled‐coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl]1/2 values (average of ⋍3.5 M) and, as well, their ΔΔGu values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea]1/2 values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the ΔΔGu values correspondingly increased with the increasing differences in electrostatic interactions (20A – 15A5R, 1.5 kcal/mol; 20A – 10A10R, 3.7 kcal/mol; and 20A – 20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the uncharged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein.
Tests for helix‐stabilizing interactions between various nonpolar side chains in alanine‐based peptides
Authors: Padmanabhan S. , Baldwin Robert L. 
Published: February 2009

AbstractStraight‐chain, non‐natural, nonpolar amino acids norleucine, norvaline, and α‐amino‐n‐butyric acid at various spacings do not interact with themselves to stabilize helix formation in alanine‐based peptides, but do interact with a Tyr spaced i, i + 4 to stabilize alanine helices, similar to the helix‐stabilizing i, i + 4 Tyr‐Leu and Tyr‐Val interactions reported earlier (Padmanabhan S, Baldwin RL, 1994, J Mol Biol 241:706–713). Leu spaced i, i + 4 from another Leu is measurably helix‐stabilizing relative to the corresponding i, i + 3 pair, but less so than for i, i + 4 Val‐Leu, Ile‐Leu, or Phe‐Leu pairs (relative to the corresponding i, i + 3 pairs) when Leu is C‐terminal to the other nonpolar amino acid. Our results indicate that limited side‐chain flexibility in an α‐helix strongly favors the interaction between 2 nonpolar residues to stabilize an isolated α‐helix.
Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp‐369 → Asn): A mechanism involving one zinc per active site
Authors: Tibbitts Thomas T. , Xu Xu , Kantrowitz Evan R. 
Published: February 2009

AbstractUsing site‐directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp‐369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000‐fold lower than the value for the wild‐type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p‐nitrophenol phosphate was 2 orders of magnitude lower than for the wild‐type enzyme. Thus the kcal/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp‐369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 Å resolution with an R‐factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X‐ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.
Redesigning the hydrophobic core of a four‐helix‐bundle protein
Authors: Munson Mary , Regan Lynne , O'Brien Ronan , Sturtevant Julian M. 
Published: February 2009

AbstractRationally redesigned variants of the 4‐helix‐bundle protein Rop are described. The novel proteins have simplified, repacked, hydrophobic cores and yet reproduce the structure and native‐like physical properties of the wild‐type protein. The repacked proteins have been characterized thermodynamically and their equilibrium and kinetic thermal and chemical unfolding properties are compared with those of wild‐type Rop. The equilibrium stability of the repacked proteins to thermal denaturation is enhanced relative to that of the wild‐type protein. The rate of chemically induced folding and unfolding of wild‐type Rop is extremely slow when compared with other small proteins. Interestingly, although the repacked proteins are more thermally stable than the wild type, their rates of chemically induced folding and unfolding are greatly increased in comparison to wild type. Perhaps as a consequence of this, their equilibrium stabilities to chemical denaturants are slightly reduced in comparison to the wild type.
Protein structural similarities predicted by a sequence‐structure compatibility method
Authors: Matsuo Yo , Nishikawa Ken 
Published: February 2009

AbstractA method for protein structure prediction has been developed, which evaluates the compatibility of an amino acid sequence with known 3‐dimensional structures and identifies the most likely structure. The method was applied to a large number of sequences in a database, and the structures of the following proteins were predicted: (1) shikimate kinase (SKase), (2) the hydrophilic subunit of mannose permease (IIABMan), (3) rat tyrosine aminotransferase (Tyr AT), and (4) threonine dehydratase (TDH). The functional and evolutionary implications of the predictions are discussed. (1) The structural similarity between SKase and adenylate kinase was predicted. Alignment of their sequences reveals that the ATP‐binding type A sequence motif and 2 ATP‐binding arginine residues are conserved. The prediction suggests a similarity in their functional mechanisms as well as an evolutionary relationship. (2) The structural similarity between IIABMan and galactose/glucose‐binding protein (GGBP) was predicted. The IIA and IIB domains are aligned with the N‐and C‐terminal domains of GGBP, respectively. The 2 phosphorylated residues, His 10 and His 175, of IIABMan are threaded onto loops located in the substrate‐binding cleft of GGBP. The prediction accounts for the phosphoryl transfer from His 10 to His 175, and to the sugar substrate. (3) The structural similarity between rat Tyr AT and Escherichia coli aspartate AT was predicted, as well as (4) the structural similarity between TDH and the tryptophan synthase β subunit. Predictions (3) and (4) support the previous predictions based on observations of the functional similarities between the proteins.
Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A2: Absence of the conserved structural water
Authors: Kumar Amarendra , Sekharudu Chandra , Ramakrishnan Boopathy , Dupureur Cynthia M. , Zhu Hongxin , Tsai Ming‐Daw , Sundaralingam Muttaiya 
Published: February 2009

AbstractTo probe the role of the Asp‐99 → His‐48 pair in phospholipase A2 (PLA2) catalysis, the X‐ray structure and kinetic characterization of the mutant Asp‐99 → Asn‐99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3121 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801–11811). The 1.9‐Å X‐ray structure of the mutant showed that the carbonyl group of Asn‐99 side chain is hydrogen bonded to His‐48 in the same way as that of Asp‐99 in the WT, thus retaining the tautomeric form of His‐48 and the function of the enzyme. The NH2 group of Asn‐99 points away from His‐48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn‐102 is hydrogen bonded to His‐57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala‐1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn‐99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala‐1.
Probing protein‐cofactor interactions in the terminal oxidases by second derivative spectroscopy: Study of bacterial enzymes with cofactor substitutions and heme a model compounds
Authors: Felsch Jason S. , Horvath Martin P. , Gursky Susan , Hobaugh Michael R. , Goudreau Paul N. , Fee James A. , Morgan William T. , Admiraal Suzanne J. , Ikeda‐Saito Masao , Fujiwara Taketomo , Fukumori Yoshihiro , Yamanaka Tateo , Copeland Robert A. 
Published: February 2009

AbstractSecond derivative absorption spectra are reported for the aa3‐cytochrome c oxidase from bovine cardiac mitochondria, the aa3‐600 ubiquinol oxidase from Bacillus subtilis, the ba3‐cytochrome c oxidase from Thermus thermophilis, and the aco‐cytochrome c oxidase from Bacillus YN‐2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450‐nm absorption band of the terminal oxidases as the 6‐coordinate low‐spin heme, cytochrome a. The spectrum of the aco‐cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6‐coordinate low‐spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine‐proline‐rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A‐(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide‐inhibited mixed‐valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm. Taken together these data suggest that the observation of Soret splitting, including a feature at 450 nm, results from specific protein‐cofactor interactions that are unique to the cytochrome a‐binding pocket of the terminal oxidases. The conservation of this unique binding pocket among evolutionarily distant species may reflect some mechanistic significance for this structure.
A “parallel plate” electrostatic model for bimolecular rate constants applied to electron transfer proteins
Authors: Watkins J. Abra , Cusanovich Michael A. , Meyer Terrance E. , Tollin Gordon 
Published: February 2009

AbstractA “parallel plate” model describing the electrostatic potential energy of protein‐protein interactions is presented that provides an analytical representation of the effect of ionic strength on a bimolecular rate constant. The model takes into account the asymmetric distribution of charge on the surface of the protein and localized charges at the site of electron transfer that are modeled as elements of a parallel plate condenser. Both monopolar and dipolar interactions are included. Examples of simple (monophasic) and complex (biphasic) ionic strength dependencies obtained from experiments with several electron transfer protein systems are presented, all of which can be accommodated by the model. The simple cases do not require the use of both monopolar and dipolar terms (i.e., they can be fit well by either alone). The biphasic dependencies can be fit only by using dipolar and monopolar terms of opposite sign, which is physically unreasonable for the molecules considered. Alternatively, the high ionic strength portion of the complex dependencies can be fit using either the monopolar term alone or the complete equation; this assumes a model in which such behavior is a consequence of electron transfer mechanisms involving changes in orientation or site of reaction as the ionic strength is varied. Based on these analyses, we conclude that the principal applications of the model presented here are to provide information about the structural properties of intermediate electron transfer complexes and to quantify comparisons between related proteins or site‐specific mutants. We also conclude that the relative contributions of monopolar and dipolar effects to protein electron transfer kinetics cannot be evaluated from experimental data by present approximations.
Effect of pH and phosphate ions on self‐association properties of the major cold‐shock protein from Bacillus subtilis
Authors: Makhatadze George I. , Marahiel Mohamed A. 
Published: February 2009

AbstractThe intermolecular interactions of the major cold‐shock protein from Bacillus subtilis (CspB) in solution in the presence of different salts, including phosphate, have been studied by means of scanning calorimetry and size‐exclusion chromatography. Calorimetric results indicate that, in all cases, protein unfolding can be approximated by a 2‐state model, but the modes of unfolding can differ depending on the conditions. In the presence of phosphate, the cooperative folding unit is a monomer, whereas in the absence of phosphate, the cooperative unit is a dimer. The difference in the self‐association of CspB in the presence and absence of phosphate was supported by size‐exclusion chromatography. These results are compared with recent structural studies of CspB in crystal and in solution.
Conformational stability of dimeric proteins: Quantitative studies by equilibrium denaturation
Authors: Neet Kenneth E. , Timm David E. 
Published: February 2009

AbstractThe conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2‐state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, δGu(H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing Gu(H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.
Six new candidate members of the α/β twisted open‐sheet family detected by sequence similarity to flavodoxin
Authors: Grandori Rita , Carey Jannette 
Published: February 2009

AbstractStrong sequence similarity has been reported among WrbA (the Trp repressor‐binding protein of Escherichia coli); Ycp4, a protein of unknown function from the budding yeast Saccharomyces cerevisiae; P25, the pap1‐dependent protein of the fission yeast Schizosaccharomyces pombe; and the translation product of a partial cDNA sequence from rice seedling root (Oryza sativa, locus Ricr02421a; here referred to as RicR). Further homology search with the profile method indicates that all the above sequences are related to the flavodoxin family and, in turn, allows detection of the recently proposed flavodoxin‐like proteins from E. coli, MioC and the hypothetical protein YihB. We discuss sequence conservation with reference to the known 3‐dimensional structures of flavodoxins. Conserved sequence and hydrophobicity patterns, as well as residue‐pair interaction potentials, strongly support the hypothesis that these proteins share the α/β twisted open‐sheet fold typical of flavodoxins, with an additional α/β unit in the WrbA family. On the basis of the proposed structural homology, we discuss the details of the putative FMN‐binding sites. Our analysis also suggests that the helix‐turn‐helix motif we identified previously in the C‐terminal region of the WrbA family is unlikely to reflect a DNA‐binding function of this new protein family.
A revised set of potentials for β‐turn formation in proteins
Authors: Hutchinson E. Gail , Thornton Janet M. 
Published: February 2009

AbstractThree thousand eight hundred ninety‐nine β‐turns have been identified and classified using a nonhomologous data set of 205 protein chains. These were used to derive β‐turn positional potentials for turn types I' and II' for the first time and to provide updated potentials for formation of the more common types I, II, and VIII. Many of the sequence preferences for each of the 4 positions in turns can be rationalized in terms of the formation of stabilizing hydrogen bonds, preferences for amino acids to adopt a particular conformation in ϕ, ψ space, and the involvement of turn types I' and II' in β‐hairpins. Only 1,632 (42%) of the turns occur in isolation; the remainder have at least 1 residue in common with another turn and have hence been classified as multiple turns. Several types of multiple turn have been identified and analyzed.
Three‐Dimensional structure of schistosoma japonicum glutathione s‐transferase fused with a six‐amino acid conserved neutralizing epitope of gp41 from hiv
Authors: Lim Kap , Ho Joseph X. , Keeling Kim , Gilliland Gary L. , Ji Xinhua , Rüker Florian , Carter Daniel C. 
Published: February 2009

AbstractThe 3‐dimensional crystal structure of glutathione S‐transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV‐1) (Muster T et al., 1993, J Virol 67:6642–6647) was determined at 2.5 Å resolution. The structure of the 3‐3 isozyme rat GST of the μ gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169–10184) was used as a molecular replacement model. The structure consists of a 4‐stranded β‐sheet and 3 α‐helices in domain 1 and 5 α‐helices in domain 2. The space group of the Sj GST crystal is P43212, with unit cell dimensions of a = b = 94.7 Å, and c = 58.1 Å. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2‐fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu‐Leu‐Asp‐Lys‐Trp‐Ala) fused to the C‐terminus of Sj GST forms a loop stabilized by symmetry‐related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes μ, α, and π. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.