bluuebunny/crossref_metadata_2025_split huggingface dataset:
The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation
Authors: Barford D. , Johnson L. N. 
Published: July 2010

AbstractThe allosteric transition of glycogen phosphorylase promoted by protein phosphorylation is accompanied by the association of a pair of functional dimers to form a tetramer. The conformational changes within the dimer that lead to the creation of a protein recognition surface have been analyzed from a comparison of the crystal structures of T‐state dimeric phosphorylase b and R‐state tetrameric phosphorylase a. Regions of the structure that participate in the tetramer interface are situated within structural subdomains. These include the glycogen storage subdomain, the C‐terminal subdomain and the tower helix. The subdomains undergo concerted conformational transitions on conversion from the T to the R state (overall r.m.s. shifts between 1 and 1.7 Å) and, together with the quaternary conformational change within the functional dimer, create the tetramer interface. The glycogen storage subdomain and the C‐terminal subdomain are distinct from those regions that contribute to the dimer interface, but shifts in the subdomains are correlated with the allosteric transitions that are mediated by the dimer interface. The structural properties of the tetramer interface are atypical of an oligomeric protein interface and are more similar to protein recognition surfaces observed in protease inhibitors and antibody‐protein antigen complexes. There is a preponderance of polar and charged residues at the tetramer interface and a high number of H‐bonds per surface area (one H‐bond per 130 Å2). In addition, the surface area made inaccessible at the interface is relatively small (1, 142 Å2 per subunit on dimer to tetramer association compared with 2,217 Å2 per subunit on monomer‐to‐dimer association).
Structural analysis of wild‐type and mutant yeast calmodulins by limited proteolysis and electrospray ionization mass spectrometry
Authors: Brockerhoff Susan E. , Edmonds Charles G. , Davis Trisha N. 
Published: July 2010

AbstractCalmodulin from Saccharomyces cerevisiae was expressed in Escherichia coli and purified. The purified protein was structurally characterized using limited proteolysis followed by ESI mass spectrometry to identify the fragments. In the presence of Ca2+, yeast calmodulin is sequentially cleaved at arginine 126, then lysine 115, and finally at lysine 77. The rapid cleavage at Arg‐126 suggests that the fourth Ca2+‐binding loop does not bind Ca2+. In the presence of EGTA, yeast calmodulin is more susceptible to proteolysis and is preferentially cleaved at Lys‐106. In addition, mutant proteins carrying I100N, E104V or both mutations, which together confer temperature sensitivity to yeast, were characterized. The mutant proteins are more susceptible than wild‐type calmodulin to proteolysis, suggesting that each mutation disrupts the structure of calmodulin. Furthermore, whereas wild‐type calmodulin is cut at Lys‐106 only in the presence of EGTA, this cleavage site is accessible in the mutants in the presence of Ca2+ as well. In these ways, the structural consequence of each mutation mimics the loss of a calcium ion in the third loop. In addition, although wild‐type calmodulin binds to four proteins in a yeast crude extract in the presence of Ca2+, the mutants bind only to a subset of these. Thus, the inability to adopt the stable Ca2+‐bound conformation in the third Ca2+‐binding loop alters the ability of calmodulin to interact with yeast proteins in a Ca2+‐dependent manner.
Guanidine derivatives restore activity to carboxypeptidase lacking arginine‐127
Authors: Phillips Margaret A. , Hedstrom Lizbeth , Rutter William J. 
Published: July 2010

AbstractArg‐127 stabilizes the oxyanion of the tetrahedral intermediate formed during Zn2+ carboxypeptidase A‐catalyzed hydrolysis. Mutant carboxypeptidases lacking Arg‐127 exhibit substantially reduced rates of hydrolysis with the change manifest almost entirely in kcat (kcat/Km is decreased by 104 for R127A). Therefore, Arg‐127 stabilizes the enzyme‐transition state complex but not the ground state enzyme‐substrate complex (Phillips, M.A., Fletterick, R., & Rutter, W.J., 1990, J. Biol. Chem. 265, 20692–20698). The addition of guanidine, methylguanidine, or ethylguanidine to R127A increases the kcat for hydrolysis of Bz‐gly‐(o)phe by 102 without changing the Km. Dissociation constants (Kd) for the guanidine derivatives range from 0.1 to 0.5 M. The binding affinity for the transition state analog Cbz‐phe‐alap(o)ala is increased similarly by 102; in contrast, the binding affinity of the ground state inhibitor benzylsuccinic acid is not altered. Thus, guanidine derivatives mimic Arg‐127 in stabilizing the rate‐limiting transition state. Hydrolysis of Bz‐gly‐(o)phe by wild‐type carboxypeptidase, R127K, or R127M is not substantially affected by guanidine derivatives. Additionally, primary amines do not change the activity of R127A. These observations imply that guanidine binds in the cavity vacated by Arg‐127 specifically and in a productive conformation for catalysis.
Spatial proximity and sequence localization of the reactive sulfhydryls of porphobilinogen synthase
Authors: Markham George D. , Myers Cynthia B. , Harris Kenneth A. , Volin Marina , Jaffe Eileen K. 
Published: July 2010

AbstractThe zinc metalloenzyme porphobilinogen synthase (PBGS) contains several functionally important, but previously unidentified, reactive sulfhydryl groups. The enzyme has been modified with the reversible sulfhydryl‐specific nitroxide spin label derivative of methyl methanethiosulfonate (MMTS), (1‐oxyl‐2,2,5,5‐tetramethyl‐Δ3‐pyrroline‐3‐methyl)methanethiosulfonate (SL‐MMTS) (Berliner, L.J., Grunwald, J., Hankovszky, H.O., & Hideg, K., 1982, Anal. Biochem. 119, 450–455). EPR spectra show that SL‐MMTS labels three groups per PBGS subunit (24 per octamer), as does MMTS. EPR signals reflecting nitroxides of different mobilities are observed. Two of the three modified cysteines have been identified as Cys‐119 and Cys‐223 by sequencing peptides produced by an Asp‐N protease digest of the modified protein. Because MMTS‐reactive thiols have been implicated as ligands to the required Zn(II), EPR spectroscopy has been used to determine the spatial proximity of the modified cysteine residues. A forbidden (Δm = 2) EPR transition is observed indicating a through‐space dipolar interaction between at least two of the nitroxides. The relative intensity of the forbidden and allowed transitions shows that at least two of the unpaired electrons are within at most 7.6 Å of each other.SL‐MMTS‐modified PBGS loses all Zn(II) and cannot catalyze product formation. The modified enzyme retains the ability to bind one of the two substrates at each active site. Binding of this substrate has no influence on the EPR spectral properties of the spin‐labeled enzyme, or on the rate of release of the nitroxides when 2‐mercaptoethanol is added. The results indicate that binding of this substrate does not affect the environment of the majority of the nitroxides. This is consistent with our previous proposal that it is the substrate that binds only to the Zn(II) enzyme that provides ligands to the catalytic Zn(II) (Jaffe, E.K., Abrams, W.R., Kaempfen, K.X., & Harris, K.A., 1992, Biochemistry 31, 2113–2123).SL‐MMTS readily forms disulfide‐linked dimers. The rate of dimer formation is proportional to the hydroxide ion concentration and is independent of both buffer and SL‐MMTS concentrations. This suggests a rate‐limiting hydrolysis of the S‐S bond of SL‐MMTS followed by rapid reaction of the liberated thiol with a second molecule of SL‐MMTS. Therefore, substantial care is required in the use of SL‐MMTS as a protein modification reagent at even slightly alkaline pH values.
Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin‐binding fibroblast growth factor receptor tyrosine kinase (flg)
Authors: Hou Jinzhao , Mckeehan Kerstin , Kan Mikio , Crabb John W. , Mckeehan Wallace L. , Carr Steven A. , Huddleston Michael J. 
Published: July 2010

AbstractFour tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin‐binding fibroblast growth factor receptor flg (FGF‐R1). Baculoviral‐insect cell‐derived recombinant FGF‐R1 was phosphorylated and fragmented with trypsin while immobilized on heparin‐agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti‐phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF‐R1 phosphorylation site. Residue 766 in the COOH‐terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.
Peptide‐protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase
Authors: Zhou Bin‐bing , Schachman H.K. 
Published: July 2010

AbstractInteraction of a 70‐amino acid zinc‐binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild‐type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N‐(phosphonacetyl)‐l‐aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183–196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50‐fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164 → Glu substitution leads to a 50‐fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508–8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231 → Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.
Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry
Authors: Carr Steven A. , Huddleston Michael J. , Bean Mark F. 
Published: July 2010

AbstractA mass spectrometry method has been developed for selective detection of glycopeptides at the low (≤25) picomole level during chromatography of glycoprotein digests and for differentiation of O‐linked from N‐linked oligosaccharides. The technique involves observation of diagnostic sugar oxonium‐ion fragments, particularly the HexNAc+ fragment at m/z 204, from collisionally excited glycopeptides. Collision‐induced fragmentation can be accomplished in either of two regions of a triple quadrupole mass spectrometer equipped with an atmospheric pressure, electrospray (ES) ionization source. If collisions before the first quadrupole are chosen, it is possible to enhance formation of carbohydrate‐related fragment ions without distorting the distribution of peptide and glycopeptide signals by increasing the collisional excitation potential only during that portion of each scan in which the low mass carbohydrate‐related ions are being detected. This procedure, requiring only a single quadrupole instrument, identifies putative glycopeptide‐containing fractions in the chromatogram but suffers from a lack of specificity in the case of co‐eluting peptides. Increased specificity is obtained by selectively detecting only those parent ions that fragment in Q2, the second collision region of the triple quadrupole, to produce an ion at m/z 204 (HexNAc+). Only (M + H)+ ions of glycopeptides are observed in these liquid chromatography‐electrospray tandem mass spectrometry (LC‐ESMS/MS) “parent‐scan” spectra. N‐linked carbohydrates are differentiated from O‐linked by LC‐ESMS/MS analysis of the digested glycoprotein prior to and after selective removal of N‐linked carbohydrates by peptide N:glycosidase F. These methods, which constitute the first liquid chromatography‐mass spectrometry (LC‐MS)‐based strategies for selective identification of glycopeptides in complex mixtures, facilitate location and preparative fractionation of glycopeptides for further structural characterization. In addition, these techniques may be used to assess the compositional heterogeneity at specific attachment sites, and to define the sequence context of the attachment site in proteins of known sequence. The strategy is demonstrated for bovine fetuin, a 42‐kDa glycoprotein containing three N‐linked, and at least three O‐linked carbohydrates. Over 90% of the fetuin protein sequence was also corroborated by these LC‐ESMS studies.
Analysis of sequence requirements for protein tyrosine sulfation
Authors: Rosenquist Grace L. , Nicholas Hugh B. 
Published: July 2010

AbstractWe analyzed sequences surrounding known tyrosine sulfation sites to determine the characteristics that distinguish these sites from those that do not undergo sulfation. Tests evaluated the number and position of acidic, basic, hydrophobic, and small amino acids, as well as disulfide and N‐glycosylation (sugar) sites. We determined that composition‐based tests that select close to 100% of known tyrosine sulfation sites reject 97% of the non‐sulfated tyrosines. The acidic test, by far the most selective, eliminated 95% of the non‐sulfated tyrosine residues and none of the sulfated tyrosines. Including the basic, hydrophobic, and disulfide tests increased the elimination rate to 97%. Whereas no position flanking the tyrosine residues had the same amino acid always present, imperfectly conserved amino acids found in some positions will improve the specificity of the tests.
Exploration of subsite binding specificity of human cathepsin D through kinetics and rule‐based molecular modeling
Authors: Scarborough Paula E. , Dunn Ben M. , Guruprasad Kunchur , Topham Chris , Blundell Tom L. , Richo Gary R. , Conner Gregory E. 
Published: July 2010

AbstractThe family of aspartic proteinases includes several human enzymes that may play roles in both physiological and pathophysiological processes. The human lysosomal aspartic proteinase cathepsin D is thought to function in the normal degradation of intracellular and endocytosed proteins but has also emerged as a prognostic indicator of breast tumor invasiveness. Presented here are results from a continuing effort to elucidate the factors that contribute to specificity of ligand binding at individual subsites within the cathepsin D active site.The synthetic peptide Lys‐Pro‐Ile‐Glu‐Phe*Nph‐Arg‐Leu has proven to be an excellent chromogenic substrate for cathepsin D yielding a value of kcat/Km = 0.92 × 10−6 s−1 M−1 for enzyme isolated from human placenta. In contrast, the peptide Lys‐Pro‐Ala‐Lys‐Phe*Nph‐Arg‐Leu and all derivatives with Ala‐Lys in the P3‐P2 positions are either not cleaved at all or cleaved with extremely poor efficiency. To explore the binding requirements of the S3 and S2 subsites of cathepsin D, a series of synthetic peptides was prepared with systematic replacements at the P2 position fixing either Ile or Ala in P3. Kinetic parameters were determined using both human placenta cathepsin D and recombinant human fibroblast cathepsin D expressed in Escherichia coli. A rule‐based structural model of human cathepsin D, constructed on the basis of known three‐dimensional structures of other aspartic proteinases, was utilized in an effort to rationalize the observed substrate selectivity.
Reconstruction of protein conformations from estimated positions of the Cα coordinates
Authors: Payne Philip W. 
Published: July 2010

AbstractProtein Cα coordinates are used to accurately reconstruct complete protein backbones and side‐chain directions. This work employs potentials of mean force to align semirigid peptide groups around the axes that connect successive Cα atoms. The algorithm works well for all residue types and secondary structure classes and is stable for imprecise Cα coordinates. Tests on known protein structures show that root mean square errors in predicted main‐chain and Cβ coordinates are usually less than 0.3 Å. These results are significantly more accurate than can be obtained from competing approaches, such as modeling of backbone conformations from structurally homologous fragments.
Conformational change of chaperone Hsc70 upon binding to a decapeptide: A circular dichroism study
Authors: Park Kyusung , Fasman Gerald D. , Flynn Gregory C. , Rothman James E. 
Published: July 2010

AbstractThe conformation of bovine Hsc70, a 70‐kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222–245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem. 203, 83–93). The result of the CD analysis of Hsc70 (15% α‐helix, 24% β‐sheet, 24% β‐turn, and 38% remainder) was very similar to the predicted secondary structure for the β‐sheet (24%) and the β‐turn (29%). However, there is disagreement between the α‐helical content by CD analysis (15%) and the predicted structure (30%). In spite of the fact that the decapeptides contained a considerable amount of β‐sheet (22%), the interaction of the heat shock protein with the peptide resulted in an overall decrease in the content of β‐sheet conformation (–15%) of the complex. This may be due to induction of a molten globule state. The result of the CCA analysis indicated that the Hsc70 undergoes a conformational change upon binding the decapeptides.
Thioflavine T interaction with synthetic Alzheimer's disease β‐amyloid peptides: Detection of amyloid aggregation in solution
Authors: Levine Harry 
Published: July 2010

AbstractThioflavine T (ThT) associates rapidly with aggregated fibrils of the synthetic β/A4‐derived peptides β(1–28) and β(1–40), giving rise to a new excitation (ex) (absorption) maximum at 450 nm and enhanced emission (em) at 482 nm, as opposed to the 385 nm (ex) and 445 nm (em) of the free dye. This change is dependent on the aggregated state as monomeric or dimeric peptides do not react, and guanidine dissociation of aggregates destroys the signal. There was no effect of high salt concentrations. Binding to the β(1–40) is of lower affinity, Kd 2 μM, while it saturates with a Kd of 0.54 μM for β(1–28). Insulin fibrils converted to a β‐sheet conformation fluoresce intensely with ThT. A variety of polyhydroxy, polyanionic, or polycationic materials fail to interact or impede interaction with the amyloid peptides. This fluorometric technique should allow the kinetic elucidation of the amyloid fibril assembly process as well as the testing of agents that might modulate their assembly or disassembly.
Structure of Paramecium tetraurelia calmodulin at 1.8 Å resolution
Authors: Rao S. T. , Wu S. , Satyshur K. A. , Sundaralingam M. , Ling K.‐Y. , Kung C. 
Published: July 2010

AbstractThe crystal structure of calmodulin (CaM; Mr 16, 700, 148 residues) from the ciliated protozoan Paramecium tetraurelia (PCaM) has been determined and refined using 1.8 Å resolution area detector data. The crystals are triclinic, space group P1, a = 29.66, b = 53.79, c = 25.49 Å, α = 92.84, β = 97.02, and γ = 88.54° with one molecule in the unit cell. Crystals of the mammalian CaM (MCaM; Babu et al., 1988) and Drosophila CaM (DCaM; Taylor et al., 1991) also belong to the same space group with very similar cell dimensions. All three CaMs have 148 residues, but there are 17 sequence changes between PCaM and MCaM and 16 changes between PCaM and DCaM. The initial difference in the molecular orientation between the PCaM and MCaM crystals was ≈︁ 7° as determined by the rotation function. The reoriented Paramecium model was extensively refitted using omit maps and refined using XPLOR. The R‐value for 11, 458 reflections with F>3σ is 0.21, and the model consists of protein atoms for residues 4–147, 4 calcium ions, and 71 solvent molecules. The root mean square (rms) deviations in the bond lengths and bond angles in the model from ideal values are 0.016 Å and 3°, respectively. The molecular orientation of the final PCaM model differs from MCaM by only 1.7°. The overall Paramecium CaM structure is very similar to the other calmodulin structures with a seven‐turn long central helix connecting the two terminal domains, each containing two Ca‐binding EF‐hand motifs. The rms deviation in the backbone N, Ca, C, and O atoms between PCaM and MCaM is 0.52 Å and between PCaM and DCaM is 0.85 Å. The long central helix regions differ, where the B‐factors are also high, particularly in PCaM and MCaM. Unlike the MCaM structure, with one kink at D80 in the middle of the linker region, and the DCaM structure, with two kinks at K75 and I85, in our PCaM structure there are no kinks in the helix; the distortion appears to be more gradually distributed over the entire helical region, which is bent with an apparent radius of curvature of 74.5(2) Å. The different distortions in the central helical region probably arise from its inherent mobility.