bluuebunny/crossref_metadata_2025_split huggingface dataset:
The amino terminus of PKA catalytic subunit—A site for introduction of posttranslation heterogeneities by deamidation: D‐Asp2 and D‐isoAsp2 containing isozymes
Authors: Kinzel Volker , König Norbert , Bossemeyer Dirk , Pipkorn Rüdiger , Lehmann Wolf D. 
Published: February 2009

AbstractConserved deamidation of PKA catalytic subunit isozymes Cα and Cβ—more than 25% at Asn2 in vivo in both cases—has been shown to yield Asp2‐ and isoAsp2‐containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7: 457–469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the “repair” of isoAsp to Asp by the action of protein L‐isoaspartyl (D‐aspartyl) O‐methyl transferase (PIMT). L‐Succinimide is prone to racemization to D‐succinimide, which may hydrolyze to D‐isoAsp‐ and D‐Asp‐containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N‐terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high‐performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp‐ and isoAsp‐residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of D‐isoAsp‐ and D‐Asp‐containing T1 fragments in addition to the L‐isomers. The small amount of the L‐isoAsp isomer, representing only part of the D‐isoAsp isomer, and the relatively large amounts of the L‐Asp and D‐Asp isomers argues for an effective action of PIMT present in cardiac tissue.
Thermodynamic and structural characterization of Asn and Ala residues in the disallowed II′ region of the Ramachandran plot
Authors: Vega M. Cristina , Serrano Luis , Martínez Jose C. 
Published: February 2009

AbstractResidue Asn47 at position L1 of a type II′ β‐turn of the α‐spectrin SH3 domain is located in a disallowed region of the Ramachandran plot (ϕ = 56 ± 12, ± = −118 ± 17). Therefore, it is expected that replacement of Asn47 by Gly should result in a considerable stabilization of the protein. Thermodynamic analysis of the N47G and N47A mutants shows that the change in free energy is small (∼0.7 kcal/mol; ∼3 kJ/mol) and comparable to that found when mutating a Gly to Ala in a α‐helix or β‐sheet. X‐ray structural analysis of these mutants shows that the conformation of the β‐turn does not change upon mutation and, therefore, that there is no relaxation of the structure, nor is there any gain or loss of interactions that could explain the small energy change. Our results indicate that the energetic definition of II' region of the Ramachandran plot (ϕ = 60 ± 30, ± = −115 ± 15) should be revised for at least Ala and Asn in structure validation and protein design.
Functional prediction: Identification of protein orthologs and paralogs
Authors: Chen Ridong , Jeong Soon‐Seog 
Published: February 2009

AbstractOrthologs typically retain the same function in the course of evolution. Using β‐decarboxylating dehydrogenase family as a model, we demonstrate that orthologs can be confidently identified. The strategy is based on our recent findings that substitutions of only a few amino acid residues in these enzymes are sufficient to exchange substrate and coenzyme specificities. Hence, the few major specificity determinants can serve as reliable markers for determining orthologous or paralogous relationships. The power of this approach has been demonstrated by correcting similarity‐based functional misassignment and discovering new genes and related pathways, and should be broadly applicable to other enzyme families.
Backbone dynamics of sequence specific recognition and binding by the yeast Pho4 bHLH domain probed by NMR
Authors: Cave John W. , Wemmer David E. , Kremer Werner 
Published: February 2009

AbstractBackbone dynamics of the basic/helix‐loop‐helix domain of Pho4 from Saccharomyces cerevisae have been probed by NMR techniques, in the absence of DNA, nonspecifically bound to DNA and bound to cognate DNA. Alpha proton chemical shift indices and nuclear Overhauser effect patterns were used to elucidate the secondary structure in these states. These secondary structures are compared to the co‐crystal complex of Pho4 bound to a cognate DNA sequence (Shimizu T, Toumoto A, Ihara K, Shimizu M, Kyogou Y, Ogawa N, Oshima Y, Hakoshima T, 1997, EMBO J 15:4689–4697). The dynamic information provides insight into the nature of this DNA binding domain as it progresses from free in solution to a specifically bound DNA complex. Relative to the unbound form, we show that formation of either the nonspecific and cognate DNA bound complexes involves a large change in conformation and backbone dynamics of the basic region. The nonspecific and cognate complexes, however, have nearly identical secondary structure and backbone dynamics. We also present evidence for conformational flexibility at a highly conserved glutamate basic region residue. These results are discussed in relation to the mechanism of sequence specific recognition and binding.
Evolution of binding affinity in a WW domain probed by phage display
Authors: Dalby Paul A. , Hoess Ronald H. , Degrado William F. 
Published: February 2009

AbstractThe WW domain is an approximately 38 residue peptide‐binding motif that binds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one‐third of its three‐stranded antiparallel β‐sheet. Improved binding to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three different concentrations of proteinase K to simultaneously drive selection for improved stability as well as high‐affinity binding. While some of the selected binders show cooperative unfolding transitions, others show noncooperative thermal unfolding curves. Two novel WW consensus sequences have been identified, which bind to the xPPxY motif with higher affinity than the wild‐type hYAP65 WW domain. These WW domain sequences are not precedented in any natural WW domain sequence. Thus, there appear to be a large number of motifs capable of recognizing the target peptide sequence, only a subset of which appear to be used in natural proteins.
Delineation of the calcineurin‐interacting region of cyclophilin B
Authors: Carpentier Mathieu , Allain Fabrice , Slomianny Marie‐Christine , Spik Geneviève , Haendler Bernard 
Published: February 2009

AbstractThe immunosuppressant drug cyclosporin A (CsA) inhibits T‐cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high‐affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin‐binding region. To delineate the calcineurin‐binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild‐type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA‐binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.
The effects of disulfide bonds on the denatured state of barnase
Authors: Clarke Jane , Hounslow Andrea M. , Fersht Alan R. , Bond Chris J. , Daggett Valerie 
Published: February 2009

AbstractThe effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended β‐sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild‐type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.
Chaperonin‐assisted folding of glutamine synthetase under nonpermissive conditions: Off‐pathway aggregation propensity does not determine the co‐chaperonin requirement
Authors: Voziyan Paul A. , Fisher Mark T. 
Published: February 2009

AbstractOne of the proposed roles of the GroEL‐GroES cavity is to provide an “infinite dilution” folding chamber where protein substrate can fold avoiding deleterious off‐pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N‐oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these “nonpermissive” folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High‐performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these “low Mg2+” conditions exist as long‐lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL‐GroES encapsulated environment is not required under all nonpermissive folding conditions.
Thermal stability of Clostridium pasteurianum rubredoxin: Deconvoluting the contributions of the metal site and the protein
Authors: Bonomi Francesco , Iametti Stefania , Mazzini Stefania , Fessas Dimitrios , Kurtz Donald M. 
Published: February 2009

AbstractTo provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 °C, and during progressive heating of the iron‐containing protein. Both methods gave a thermal stability of metal binding in the order Fe2+ « Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 °C for Fe2+Rd and 83 °C for Fe3+Rd. Temperature‐dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and 1H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50‐70 °C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower‐temperature structural changes is dependent on the size of the MS4 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M‐SCys bond strength. In the case of Fe3+Rd, stoichiometric Fe3+/cysteine‐ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)4 site.
Fibers of tau fragments, but not full length tau, exhibit a cross β‐structure: Implications for the formation of paired helical filaments
Authors: Giannetti A. M. , Lindwall G. , Kohlstaedt L. A. , Chau M.‐F. , Radeke M. J. , Feinstein S. C. 
Published: February 2009

AbstractWe have used X‐ray fiber diffraction to probe the structure of fibers of tau and tau fragments. Fibers of fragments from the microtubule binding domain had a cross β‐structure that closely resembles that reported both for neurofibrillary tangles found in Alzheimer's disease brain and for fibrous lesions from other protein folding diseases. In contrast, fibers of full‐length tau had a different, more complex structure. Despite major differences at the molecular level, all fiber types exhibited very similar morphology by electron microscopy. These results have a number of implications for understanding the etiology of Alzheimer's and other tauopathic diseases. The morphology of the peptide fibers suggests that the region in tau corresponding to the peptides plays a critical role in the nucleation of fiber assembly. The dramatically different structure of the full length tau fibers suggests that some region in tau has enough inherent structure to interfere with the formation of cross β‐fibers. Additionally, the similar appearance by electron microscopy of fibrils with varying molecular structure suggests that different molecular arrangements may exist in other samples of fibers formed from tau.
Characterization of the interface structure of enzyme‐inhibitor complex by using hydrogen‐deuterium exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry
Authors: Akashi Satoko , Takio Koji 
Published: February 2009

AbstractWe investigated the interaction between a thiol protease inhibitor, cystatin, and its target enzyme, papain, by hydrogen‐deuterium (H/D) exchange in conjunction with successive analysis by collision‐induced dissociation (CID) in an rf‐only hexapole ion guide with electrospray ionization‐Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain, examining the mass of each fragment produced by hexapole‐CID. In the absence of papain, amide hydrogens in short amino‐terminal fragments, such as b2+10 and b2+12, were highly deuterated within 1 min. Although fewer fragments were observed for the cystatin‐papain complex in the hexapole‐CID spectra, significant reductions in initial deuterium content were recognized throughout the sequence of cystatin. This suggests that complex formation restricted the flexibility of the whole cystatin molecule. Detailed analyses revealed that a marked reduction in deuterium content in the region of residues 1‐10 persisted for hours, suggesting that the flexible N‐terminal region was tightly fixed in the binding pocket with hydrogen bonds. Our results are consistent with those of previous studies on the structure and inhibition mechanism of cystatin. We demonstrated here that enzyme‐inhibitor interactions can be characterized by H/D exchange in combination with CID in a hexapole ion guide using ESI‐FTICR MS rapidly and using only a small amount of sample.
For the record: Effect of deamidation on folding of ribonuclease A
Authors: Orrù Stefania , Vitagliano Luigi , Esposito Luciana , Mazzarella Lelio , Marino Gennaro , Ruoppolo Margherita 
Published: February 2009

AbstractThe folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65‐72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn → isoAsp where the local structure of the loop 65‐72 has been modified keeping intact the C65‐C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild‐type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65‐72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.
Characterization of an anti‐Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody‐bound antigen and mass spectrometricpeptide mapping
Authors: Legros Vèronique , Jolivet‐Reynaud Colette , Battail‐Poirot Nicole , Saint‐Pierre Christine , Forest Eric 
Published: February 2009

AbstractLyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.
Nonpolar contributions to conformational specificity in assemblies of designed short helical peptides
Authors: Boon Chandra L. , Chakrabartty Avijit 
Published: February 2009

AbstractA series of designed short helical peptides was used to study the effect of nonpolar interactions on conformational specificity. The consensus sequence was designed to obtain short helices (17 residues) and to minimize the presence of interhelical polar interactions. Furthermore, the sequence contained a heptad repeat (abcdefg), where positions a and d were occupied by hydrophobic residues Leu, Ile, or Val, and positions e and g were occupied by Ala. The peptides were named according to the identities of the residues in the adeg positions, respectively. The peptides llaa, liaa, ilaa, iiaa, ivaa, viaa, lvaa, vlaa, and vvaa were synthesized, and their characterization revealed marked differences in specificity. An experimental methodology was developed to study the nine peptides and their pairwise mixtures. These peptides and their mixtures formed a vast array of structural states, which may be classified as follows: helical tetramers and pentamers, soluble and insoluble helical aggregates, insoluble unstructured aggregates, and soluble unstructured monomers. The peptide liaa formed stable helical pentamers, and iiaa and vlaa formed stable helical tetramers. Disulfide cross‐linking experiments indicated the presence of an antiparallel helix alignment in the helical pentamers and tetramers. Rates of amide proton exchange of the tetrameric form of vlaa were 10‐fold slower than the calculated exchange rate for unfolded vlaa. In other work, the control of specificity has been attributed to polar interactions, especially buried polar interactions; this work demonstrated that subtle changes in the configuration of nonpolar interactions resulted in a large variation in the extent of conformational specificity of assemblies of designed short helical peptides. Thus, nonpolar interactions can have a significant effect on the conformational specificity of oligomeric short helices.
For the record: Temperature‐sensitive suppressor mutations of the Escherichia coli DNA gyrase B protein
Authors: Blance Stephen J. , Williams Nicola L. , Preston ZOË A. , Bishara John , Smyth Michael S. , Maxwell Anthony 
Published: February 2009

AbstractEscherichia coli strain LE316 contains a mutation in gyrB that results in the substitution of Val164 to Gly and confers both chlorobiocin resistance and temperature sensitivity. Selection for suppressors of the ts phenotype yielded second‐site mutations in GyrB at His38 and Thr157. The properties of proteins bearing these mutations have been characterized, and a mechanism of suppression is proposed based upon structural considerations.
NMR characterization of a pH‐dependent equilibrium between two folded solution conformations of the pheromone‐binding protein from Bombyx mori
Authors: Damberger Fred , Horst Reto , Wüthrich Kurt , Peng Guihong , Nikonova Larisa , Leal Walter Soares 
Published: February 2009

AbstractNMR spectroscopic changes as a function of pH in solutions of the pheromone‐binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single “acid form” (A), and a homogeneous “basic form” (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a welldispersed NMR spectrum, indicating that it represents a more structured, “closed” conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant‐binding proteins, and it will be of interest to further investigate possible correlations with pH‐dependent regulation of ligand binding and release in the biological function of this class of proteins.