bluuebunny/crossref_metadata_2025 huggingface dataset:
Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins
Authors: Bihoreau M.T. , Baudin V. , Marden M. , Lacaze N. , Bohn B. , Kister J. , Poyart C. , Pagnier J. , Schaad O. , Edelstein S.J. , Dumoulin A.
Published: July 2010

Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb S, β6 Glu → Val) have been obtained from X‐ray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Site‐directed mutagenesis with expression of the Hb S β subunits in Escherichia coli provides the experimental tools to test these models. For Hb S, the β6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88. Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydropho‐bicity of the residue at the β6 position with Ile > Val > Ala. We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the β36 location. Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation. The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact. Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand. This is demonstrated by associating the β23 Val → Ile mutation at the axial contact with either the β6 Glu → Val or β6 Glu → Ile substitution in the same β subunit. In comparison to native Hb S the solubility of these deoxyHb double mutants is decreased two‐ and fourfold, respectively.
Calculation of the free energy of association for protein complexes
Authors: Horton Nancy , Lewis Mitchell
Published: July 2010

We have developed a method for calculating the association energy of quaternary complexes starting from their atomic coordinates. The association energy is described as the sum of two solvation terms and an energy term to account for the loss of translational and rotational entropy. The calculated solvation energy, using atomic solvation parameters and the solvent accessible surface areas, has a correlation of 96% with experimentally determined values. We have applied this methodology to examine intermediates in viral assembly and to assess the contribution isomerization makes to the association energy of molecular complexes. In addition, we have shown that the calculated association can be used as a predictive tool for analyzing modeled molecular complexes.
Reconstructing history with amino acid sequences1
Authors: Doolittle Russell F.
Published: July 2010

The main goal of the protein evolutionist is the reconstruction of past events leading to the structures of contemporary proteins. The common strategy is to align amino acid sequences and make inferences about matters of common ancestry. The rate of change of amino acid sequence varies greatly from protein to protein, and this naturally affects how far back a given protein's ancestry can be traced. Happily, the rate of change of many proteins is slow enough that very ancient events can be inferred. Many mainstream metabolic enzymes, for example, are 40–50% identical in prokaryotes and eukaryotes, groups that diverged from a common ancestor more than 1.5 billion years ago. Moreover, some eukaryotic proteins like actin and tubulin change so slowly that they are seldom less than 60% identical, no matter from what source they are drawn. As it happens, prokaryotic counterparts for many eukaryotic cytoskeletal proteins are unknown. A recent exception involves the finding that a heat shock protein cognate is a relative of actin. The gene duplication that gave rise to these two proteins must have been an ancient event. The more recent invention of other proteins whose distribution is restricted to one or the other of the major kingdoms may be easier to trace. Among the factors that can confound the reconstruction of events, however, are occasional horizontal gene transfers and exon shuffling. The latter has led to a number of mosaic proteins, many of which contain various combinations of a relatively small set of modules like the epidermal growth factor domain.
Atomic solvation parameters applied to molecular dynamics of proteins in solution
Authors: Wesson Laura , Eisenberg David
Published: July 2010

A solvation energy function for use in the molecular simulation of proteins is proposed. It is based on the accessible surface areas of atoms in the protein and on atomic solvation parameters derived from empirical vapor‐to‐water free energies of transfer of amino acid side‐chain analogs. The energy function and its derivatives were added to the CHARMM molecular simulation program (Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S.,&Karplus, M., 1983, J. Comput. Chem. 4(2), 187–217). The effect of the added energy term was evaluated by 110 ps of molecular dynamics on the 26‐residue protein melittin. The melittin monomer and tetramer were studied both with and without the added term. With the added energy term the monomer partially unfolded, while the secondary structure of the tetramer was preserved, in agreement with reported experiments (Brown, L.R., Lauterwein, J.,&Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 231–244; Lauterwein, J., Brown, L.R.,&Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 219–230).
Characterization, primary structure, and evolution of lamprey plasma albumin
Authors: Gray Jeffrey E. , Doolittle Russell F.
Published: July 2010

The most abundant protein found in blood plasma from the sea lamprey (Petromyzon marinus) has the hallmarks of a plasma albumin: namely, high abundance, solubility in distilled water, a small number of tryptophans, and a high content of cysteines and charged residues. As in other vertebrate albumins, not all the cysteines are disulfide bonded. An unusual feature of this protein is its molecular weight of 175,000, roughly 2.5 times the size of other vertebrate albumins. Its amino acid sequence, deduced from a series of overlapping cDNA clones, can be aligned with other members of the gene family including plasma albumin, alpha‐fetoprotein, and vitamin‐D binding protein, confirming that it is indeed an oversized albumin. An unusual feature of the sequence is a 28‐amino acid stretch consisting of a serine‐threonine repeat with the general motif (STTT). Lamprey albumin contains a 23‐amino acid putative signal peptide and a 6‐residue putative propeptide, which, when cleaved, yield a mature protein of 1,394 amino acids with a calculated molecular weight of 157,000. The sequence also includes nine potential N‐linked glycosylation sites (Asn‐X‐Ser/Thr), consistent with observation that lamprey albumin is a glycoprotein. If all the potential glycosylation sites were occupied by clusters of 2,000 molecular weight each, the total molecular weight would be 175,000. Like other members of the gene family, lamprey albumin is composed of a series of 190‐amino acid repeats, there being seven such domains all together. Quantitative amino acid sequence comparisons of lamprey albumin with the other members of the gene family indicate that it diverged from an ancestral albumin prior to the gene duplications leading to this diverse group. This notion is confirmed by the pattern of amino acid insertions and deletions observed in a consideration of all domains that compose this family. Furthermore, it suggests that the invention of albumin antedates the vertebrate radiation.
Lysine‐21 of Leuconostoc mesenteroides glucose 6‐phosphate dehydrogenase participates in substrate binding through charge–charge interaction
Authors: Lee W. Theodore , Levy H. Richard
Published: July 2010

Leuconostoc mesenteroides glucose 6‐phosphate dehydrogenase (G6PD) was isolated in high yield and purified to homogeneity from a newly constructed strain of Escherichia coli which lacks its own glucose 6‐phosphate dehydrogenase gene. Lys‐21 is one of two lysyl residues in the enzyme previously modified by the affinity labels pyridoxal 5′‐phosphate and pyridoxal 5′‐diphosphate‐5′‐adenosine, which are competitive inhibitors of the enzyme with respect to glucose 6‐phosphate (LaDine, J.R., Carlow, D., Lee, W.T., Cross, R.L., Flynn, T.G., & Levy, H.R., 1991, J. Biol. Chem. 266, 5558–5562). K21R and K21Q mutants of the enzyme were purified to homogeneity and characterized kinetically to determine the function of Lys‐21. Both mutant enzymes showed increased Km‐values for glucose 6‐phosphate compared to wild‐type enzyme: 1.4‐fold (NAD‐linked reaction) and 2.1‐fold (NADP‐linked reaction) for the K21R enzyme, and 36‐fold (NAD‐linked reaction) and 53‐fold (NADP‐linked reaction) for the K21Q enzyme. The Km for NADP+ was unchanged in both mutant enzymes. The Km for NAD+ was increased 1.5‐ and 3.2‐fold, compared to the wild‐type enzyme, in the K21R and K21Q enzymes, respectively. For the K21R enzyme the kcat
for the NAD‐ and NADP‐linked reactions was unchanged. The kcat for the K21Q enzyme was increased in the NAD‐linked reaction by 26% and decreased by 30% in the NADP‐linked reaction from the values for the wild‐type enzyme. The data are consistent with Lys‐21 participating in the binding of the phosphate group of the substrate to the enzyme via charge–charge interaction.
The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation
Authors: Barford D. , Johnson L. N.
Published: July 2010

The 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

Calmodulin 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

Arg‐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

The 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

Four 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

Interaction 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

A 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

We 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

The 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

Protein 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

The 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

Thioflavine 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

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

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

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

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

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

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

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

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

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

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

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