bluuebunny/crossref_metadata_2025_split huggingface dataset:
The mycobacterium tuberculosis small heat shock protein Hsp16.3 exposes hydrophobic surfaces at mild conditions: Conformational flexibility and molecular chaperone activity
Authors: Yang Hongmei , Huang Sufang , Dai Hongzheng , Gong Yandao , Zheng Changxue , Chang Zengyi 
Published: October 2010

AbstractHsp16.3, the α‐crystallin‐related small heat shock protein of Mycobacterium tuberculosis that is maximally expressed during the stationary phase and is a major membrane protein, has been reported to form specific trimer‐of‐trimers structure and to act as an effective molecular chaperone (Chang Z et al., 1996, J Biol Chem 271:7218—7223). However, little is known about its action mechanism. In this study, Hsp16.3 conformational intermediates with dramatically increased chaperone activities were detected after treatment with very low concentrations of guanidine hydrochloride (0.05 M), urea (0.3 M), or mild heating (30 8C). The intermediates showed a significant increase in their capacity to bind the hydrophobic probe 1‐anilino‐8‐naphthalene sulfonate (ANS), indicating an increased exposure of hydrophobic surfaces. Interestingly, the greatest chaperone activities of Hsp16.3 were observed in the presence of 0.3 M guanidine HCl or when heated to 35 °C. CD spectroscopy studies revealed no significant changes in protein secondary and tertiary structures at these mild treatments. Our in vitro studies also indicate that long‐time‐heated Hsp16.3, heated even to temperatures as high as 85 °C, has almost the same, if not a slightly greater, chaperone activities as the native protein when cooled to room temperature and its secondary structures also almost recovered. Together, these results suggest that Hsp16.3 modulates its chaperone activity by exposing hydrophobic surfaces and that the protein structure is highly stable and flexible, thus highly adapted for its function.
The influence of C‐terminal extension on the structure of the “J‐domain” in E. coli DnaJ
Authors: Huang Kai , Flanagan John M. , Prestegard James H. 
Published: October 2010

AbstractTwo different recombinant constructs of the N‐terminal domain in Escherichia coli DnaJ were uniformly labeled with nitrogen‐15 and carbon‐13. One, DnaJ(1—78), contains the complete “J‐domain,” and the other, DnaJ(1—104), contains both the “J‐domain” and a conserved “G/F” extension at the C‐terminus. The three‐dimensional structures of these proteins have been determined by heteronuclear NMR experiments. In both proteins the “J‐domain” adopts a compact structure consisting of a helix‐turn‐helix‐loop‐helix‐turn‐helix motif. In contrast, the “G/F” region in DnaJ(1—104) does not fold into a well‐defined structure. Nevertheless, the “G/F” region has been found to have an effect on the packing of the helices in the “J‐domain” in DnaJ(1—104). Particularly, the interhelical angles between Helix IV and other helices are significantly different in the two structures. In addition, there are some local conformational changes in the loop region connecting the two central helices. These structural differences in the “J‐domain” in the presence of the “G/F” region may be related to the observation that DnaJ(1—78) is incapable of stimulating the ATPase activity of the molecular chaperone protein DnaK despite evidence that sites mediating the binding of DnaJ to DnaK are located in the 1—78 segment.
Stereoselectivity of mucorales lipases toward triradylglycerols—A simple solution to a complex problem
Authors: Scheib Holger , Pleiss Jürgen , Schmid Rolf D. , Kovac Andrea , Paltauf Fritz 
Published: October 2010

AbstractThe lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn‐2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O'‐C1′ ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N′‐C1′ amide bond or a phenyl ring in sn‐2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn‐1 and sn‐3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn‐1. To explain these experimental observations, computer‐aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C‐terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn‐1 and sn‐3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity.
The insect immune protein scolexin is a novel serine proteinase homolog
Authors: Finnerty Casey M. , Granados Robert R. , Karplus P. Andrew 
Published: October 2010

AbstractScolexin is a coagulation‐provoking plasma protein induced in response to bacterial or viral infection of larval Manduca sexta, a large lepidopterous insect. Here we report the isolation and sequencing of two cDNA clones that code for scolexin isoforms sharing 80% sequence identity. The scolexin sequences have low but recognizable sequence similarity to members of the chymotrypsin family and represent a new subfamily of chymotrypsin‐like serine proteinases. Comparison with known structures reveals the conservation of key catalytic residues and a possible specificity for small nonpolar residues. Most remarkable is the absence of a canonical activation peptide cleavage site. This suggests that the regulation of scolexin activity will involve a novel activation mechanism.
For the record: Structural consequences of the active site substitution Cys181 → Ser in metallo‐β‐lactamase from bacteroides fragilis
Authors: Li Zhong , Herzberg Osnat , Rasmussen Beth A. 
Published: October 2010

AbstractThe metallo‐β‐lactamases require divalent cations such as zinc or cadmium for hydrolyzing the amide bond of β‐lactam antibiotics. The crystal structure of the Zn2+‐bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the β‐lactam bond of the substrate. It was previously reported that the replacement of the active site Cys181 by a serine residue severely impaired catalysis while atomic absorption measurements indicated that binding of the two zinc ions remained intact. Contradicting data emerge from recent mass spectrometry results, which show that only a single zinc ion binds to the C181S metallo‐β‐lactamase. In the current study, the C181S mutant enzyme was examined at the atomic level by determining the crystal structure at 2.6 Å resolution. The overall structure of the mutant enzyme is the same as that of the wild‐type enzyme. At the mutation site, the side chain of Ser181 occupies the same position as that of the side chain of Cys181 in the wild‐type protein. One zinc ion, Zn1, is present in the crystal structure; however, the site of the second zinc ion, Zn2 is unoccupied. A water molecule is associated with Zn1, reminiscent of the hydroxide seen in the structure of the wild‐type enzyme but farther from the metal. The position of the water molecule is off the plane of the carboxylate group of Asp103; therefore, the water molecule may be less nucleophilic than a water molecule which is coplanar with the carboxylate group.
Quench‐flow experiments combined with mass spectrometry show apomyoglobin folds through an obligatory intermediate
Authors: Tsui Vickie , Garcia Carlos , Cavagnero Silvia , Siuzdak Gary , Dyson H. Jane , Wright Peter E. 
Published: October 2010

AbstractFolding of apomyoglobin is characterized by formation of a compact intermediate that contains substantial helicity. To determine whether this intermediate is obligatory or whether the protein can fold directly into the native state via an alternate parallel pathway, we have combined quench‐flow hydrogen‐exchange pulse labeling techniques with electrospray ionization mass spectrometry. The mass spectra of apomyoglobin obtained at various refolding times suggest that apomyoglobin indeed folds through a single pathway containing an obligatory intermediate with a significant hydrogen‐bonded secondary structure content.
Functional implications of structural differences between variants A and B of bovine β‐lactoglobulin
Authors: Qin Bin Y. , Jameson Geoffrey B. , Bewley Maria C. , Baker Edward N. , Creamer Lawrence K. 
Published: October 2010

AbstractThe structure of the trigonal crystal form of bovine β‐lactoglobulin variant B at pH 7.1 has been determined by X‐ray diffraction methods at a resolution of 2.22 Å and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine β‐lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014—14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61—67), there are small changes in main‐chain conformation, whereas the substitution V118A on β‐strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH‐dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.
Crystal structure of a designed, thermostable, heterotrimeric coiled coil
Authors: Nautiyal Shivani , Alber Tom 
Published: October 2010

AbstractElectrostatic interactions are often critical for determining the specificity of protein‐protein complexes. To study the role of electrostatic interactions for assembly of helical bundles, we previously designed a thermostable, heterotrimeric coiled coil, ABC, in which charged residues were employed to drive preferential association of three distinct, 34‐residue helices. To investigate the basis for heterotrimer specificity, we have used multiwavelength anomalous diffraction (MAD) analysis to determine the 1.8 Å resolution crystal structure of ABC. The structure shows that ABC forms a heterotrimeric coiled coil with the intended arrangement of parallel chains. Over half of the ion pairs engineered to restrict helix associations were apparent in the experimental electron density map. As seen in other trimeric coiled coils, ABC displays acute knobs‐into‐holes packing and a buried anion coordinated by core polar amino acids. These interactions validate the design strategy and illustrate how packing and polar contacts determine structural uniqueness.
Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response innicotiana sylvestris
Authors: Toedt John M. , Braswell Emory H. , Schuster Todd M. , Yphantis David A. , Taraporewala Zenobia F. , Culver James N. 
Published: October 2010

AbstractThe hypersensitivity resistance response directed by the N' gene inNicotiana sylvestrisis elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild‐type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild‐type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4°C. Viral reconstitution kinetics at 20°C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20°C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allowsN. sylvestristo recognize the invading virus.
Crystal structure of wild‐type human procathepsin K
Authors: Sivaraman J. , Lalumière1 Manon , Ménard Robert , Cygler Miroslaw 
Published: October 2010

AbstractCathepsin K is a lysosomal cysteine protease belonging to the papain superfamily. It has been implicated as a major mediator of osteoclastic bone resorption. Wild‐type human procathepsin K has been crystallized in a glycosylated and a deglycosylated form. The latter crystals diffract better, to 3.2 å resolution, and contain four molecules in the asymmetric unit. The structure was solved by molecular replacement and refined to an R‐factor of 0.194. The N‐terminal fragment of the proregion forms a globular domain while the C‐terminal segment is extended and shows substantial flexibility. The proregion interacts with the enzyme along the substrate binding groove and along the proregion binding loop (residues Ser138–Asn156). It binds to the active site in the opposite direction to that of natural substrates. The overall binding mode of the proregion to cathepsin K is similar to that observed in cathepsin L, caricain, and cathepsin B, but there are local differences that likely contribute to the specificity of these proregions for their cognate enzymes. The main observed difference is in the position of the short helix α3p (67p–75p), which occupies the S' subsites. As in the other proenzymes, the proregion utilizes the S2 subsite for anchoring by placing a leucine side chain there, according to the specificity of cathepsin K toward its substrate.
Comparison of the backbone dynamics of the apo‐ and holo‐carboxy‐terminal domain of the biotin carboxy1 carrier subunit of Escherichia coli acety1‐CoA carboxylase
Authors: Yao Xiang , Soden Cylburn , Summers Michael F. , Beckett Dorothy 
Published: October 2010

AbstractThe biotin carboxyl carrier protein (BCCP) is a subunit of acetyl‐CoA carboxylase, a biotin‐dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein–protein interactions with three distinct partners, depending on its state of post‐translational modification. Apo‐BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post‐translational attachment of biotin to a single lysine residue on BCCP. Holo‐BCCP then interacts with the biotin carboxylase subunit of acetyl‐CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxy‐biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl‐CoA to form malonyl‐CoA. The determinants of protein–protein interaction specificity in this system are unknown. The NMR solution structure of the unbiotinylated form of an 87 residue C‐terminal domain fragment (residue 70–156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C‐terminal fragment (residue 77–156) of BCCP from Escherichia coli acetyl‐CoA carboxylase have previously been determined. Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin‐binding region upon biotinylation of the protein. These structural changes may be important for regulating specific protein–protein interactions. Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein. The results indicate that upon biotinylation, the inherent mobility of the biotin‐binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.
Tolerance of a protein to multiple polar‐to‐hydrophobic surface substitutions
Authors: Cordes Matthew H.J. , Sauer Robert T. 
Published: October 2010

AbstractHydrophobic substitutions at solvent‐exposed positions in two α‐helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild‐type Arc, with a rough correlation between the number of polar‐to‐hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar‐to‐hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a tm 6°C higher than wild‐type. By contrast, a helix A mutant with three similar polar‐to‐hydrophobic substitutions was 23°C less stable than wild‐type. Also, one set of three polar‐to‐hydrophobic substitutions in helix B was tolerated when introduced into the wild‐type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues.
Topology and dynamics of the 10 kDa C‐terminal domain of DnaK in solution
Authors: Bertelsen Eric B. , Zhou Hongjun , Lowry David F. , Flynn Gregory C. , Dahlquist Frederick W. 
Published: October 2010

AbstractHsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N‐terminal ATPase domain, a 17 kDa peptide‐binding domain, and a 10 kDa C‐terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C‐terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C‐terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high‐resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C‐terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full‐length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle‐like structure in solution.
Is human thioredoxin monomeric or dimeric?
Authors: Gronenborn Angela M. , Clore1 G. Marius , Louis John M. , Wingfield Paul T. 
Published: October 2010

AbstractWe have examined the molecular weight and rotational correlation time of human thioredoxin by analytical ultracentrifugation and NMR spectroscopy, respectively. Two variants of human thioredoxin were studied, namely human thioredoxin identical in amino acid sequence to the one whose NMR structure we previously determined (C62A, C69A, C73A, M74T) and human thioredoxin (C62A, C69A, C73A, M74) containing the wild‐type amino acid methionine at position 74. In both cases, the experimental data indicate that the predominant species is monomeric and we find no evidence for the existence of a well‐defined dimeric form as was observed in the recently reported crystal structure (Weichsel et al., 1996) of human thioredoxin and the C73S mutant.
Variants of ribonuclease inhibitor that resist oxidation
Authors: Kim Byung‐Moon , Schultz L.Wayne , Raines Ronald T. 
Published: October 2010

AbstractHuman ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic‐type ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight‐membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10‐to 15‐fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328–Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild‐type hRI for inhibiting pancreatic‐type ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein.
Expression and characterization of the intact N‐terminal domain of streptokinase
Authors: Azuaga Ana I. , Woodruff Nicholas D. , Conejero‐Lara Francisco , Cox Vivienne F. , Smith Richard A.G. , Dobson Christopher M. 
Published: October 2010

AbstractProteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero‐Lara F et al., 1996, Protein Sci 5:2583–2591). The N‐terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage. To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N‐terminal region of the protein, residues 1–146. The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein.