bluuebunny/crossref_metadata_2025_split huggingface dataset:
Taxonomy and function of C1 protein kinase C homology domains
Authors: Hurley James H. , Newton Alexandra C. , Parker Peter J. , Blumberg Peter M. , Nishizuka Yasutomi 
Published: July 2010

AbstractC1 domains are compact α/β structural units of about 50 amino acids which tightly bind two zinc ions. These domains were first discovered as the loci of phorbol ester and diacylglycerol binding to conventional protein kinase C isozymes, which contain two C1 domains (CIA and C1B) in their N‐terminal regulatory regions. We present a comprehensive list of 54 C1 domains occurring singly or doubly in 34 different proteins. Many C1 domains and C1 domain‐containing proteins bind phorbol esters, but many others do not. By combining analysis of 54 C1 domain sequences with information from previously reported solution and crystal structure determinations and site‐directed mutagenesis, profiles are derived and used to classify C1 domains. Twenty‐six C1 domains fit the profile for phorbol‐ester binding and are termed “typical.” Twenty‐eight other domains fit the profile for the overall C1 domain fold but do not fit the profile for phorbol ester binding, and are termed “atypical.” Proteins containing typical C1 domains are predicted to be regulated by diacylglycerol, whereas those containing only atypical domains are not.
Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals
Authors: Mozzarelli Andrea , Rivetti Claudio , Rossi Gian Luigi , Eaton William A. , Henry Eric R. 
Published: July 2010

AbstractIn solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of G‐state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 52:2888–2906) suggested that there are high‐ and low‐affinity subunit conformations of G, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of G‐state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the “strong” allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10‐30‐fold. We find no change in affinity (<10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two‐state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88–118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the α and β subunits. This was shown by calculating the separate α and β subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X‐ray structures of deoxygenated and fully oxygenated T‐state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775–792).
Structural comparison of cupredoxin domains: Domain recycling to construct proteins with novel functions
Authors: Murphy MICHAEL E. P. , Lindley Peter F. , Adman Elinor T. 
Published: July 2010

AbstractThe three‐dimensional structures of the copper‐containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor. The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins. Simultaneous structural superposition of the enzyme domains and their known cupredoxin relatives shows clearly that there are at least six cupredoxin classes, and that the evolution of the conserved core of these domains is independent of the presence or absence of copper sites. Relationships among the variable loops in these structures show that the two‐domain ancestor of the blue oxidases contained a trinuclear‐copper interface but could not have functioned in a monomeric state. Comparison of the sequence of the copper‐containing, iron‐regulating protein. Ferrous transport (Fet3) from yeast to the structurally defined core and loop residues of the cupredoxins suggests specific residues that could be involved in the ferroxidase activity of Fet3.
Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X‐ray crystallography
Authors: Su Shaoyu , Gao Yi‐Gui , Zhang Hong , Terwilliger Thomas C. , Wang ANDREW H.‐J. 
Published: July 2010

AbstractThe high‐resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, f1, fd) has been solved recently for the wild‐type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP‐ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4:187–197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer‐dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X‐ray crystallographic analysis at 2.0 Å resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long‐range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.
Intermolecular tuning of calmodulin by target peptides and proteins: Differential effects on Ca2+ binding and implications for kinase activation
Authors: Peersen Olve B. , Madsen Travis S. , Falke Joseph J. 
Published: July 2010

AbstractCa2+‐activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM‐regulated proteins: phosphorylase kinase, CaM‐dependent protein kinase 11, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca2+‐ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N‐terminal regulatory domain from 14‐ to 350‐fold and slow its Ca2+ dissociation kinetics from 60‐ to 140‐fold. Smaller effects are observed for the C‐terminal domain, where peptides increase the apparent Ca2+ affinity 8‐ to 100‐fold and slow dissociation kinetics 13‐ to 32‐fold. In full‐length skeletal myosin light chain kinase the inter‐molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM‐protein complex that has a 10‐fold lower Ca2+ affinity than the analogous CaM‐peptide complex. Unlike the CaM‐peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N‐terminal domain are substantially occluded in the CaM‐protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free CaZ2+‐CaM or Cq2+‐CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate‐limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C‐terminal domain or the dissociation of Ca2+ loaded C‐terminal domain from skMLCK. The observed target‐induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.
Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol
Authors: De Laureto Patrizia Polverino , Scaramella Elena , De Filippis Vincenzo , Bruix Marta , Rico Manuel , Fontana Angelo 
Published: July 2010

AbstractWe have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE‐resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124‐residue chain of RNase in its TFE state (20–42 °C, 6–24 h) at peptide bond Asn 34‐Leu 35, followed by a slower cleavage at peptide bond Thr 45‐Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1–34 and 35–124) (RNase Th1) or three (1–34, 35–45 and 46–124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Th1 maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (‐ΔTm 16 °C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Th1 is closely similar to that of the intact protein was confirmed unambiguously by two‐dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30–41, i.e., at residues nearby the cleaved Asn 34‐Leu 35 peptide bond. RNase Th1 retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 31–39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41–48 region forms a β‐strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Th1 and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE‐state that likely dictate the selective proteolysis phenomenon by thermolysin.
Characterization and crystallization of a minimal catalytic core domain from mammalian type II adenylyl cyclase
Authors: Zhang Gongyi , Liu Yu , Qin Jun , Vo Binh , Tang Wei‐Jen , Ruoho Arnold E. , Hurley James H. 
Published: July 2010

AbstractAdenylyl cyclases play a pivotal role in signal transduction by carrying out the regulated synthesis of cyclic AMP. The nine cloned mammalian adenylyl cyclases all share two conserved regions of sequence, C1and C2, which are homologous to each other and are together responsible for catalytic activity. Recombinant C1and C2domains catalyze the synthesis of cyclic AMP when they are mixed and activated by forskolin, and C2domains alone also manifest reduced levels of forskolin‐stimulated enzyme activity. Using limited proteolysis and mass spectrometry, we have mapped the boundaries of a minimal stable and active C2catalytic domain to residues 871–1090 of type II adenylyl cyclase. We report the properties and crystallization of this trimmed domain, termed IIC2‐Δ4. Crystals belong to space group P4n212, wheren= 1 or 3;a=b= 81.3, andc= 180.5 Å; and there are two molecules per asymmetric unit related by an approximate body centering operation. Flash‐frozen crystals diffract anisotropically to 2.2 Å along thec*direction and to 2.8 Å along thea*andb*directions using synchrotron radiation.
How do potentials derived from structural databases relate to “true” potentials?
Authors: Zhang Li , Skolnick Jeffrey 
Published: July 2010

AbstractKnowledge‐based potentials are used widely in protein folding and inverse folding algorithms. Two kinds of derivation methods are used. (1) The interactions in a database of known protein structures are assumed to obey a Boltzmann distribution. (2) The stability of the native folds relative to a manifold of misfolded structures is optimized. Here, a set of previously derived contact and secondary structure propensity potentials, taken as the “true” potentials, are employed to construct an artificial protein structural database from protein fragments. Then, new sets of potentials are derived to see how they are related to the true potentials. Using the Boltzmann distribution method, when the stability of the structures in the database lies within a certain range, both contact potentials and secondary structure propensities can be derived separately with remarkable accuracy. In general, the optimization method was found to be less accurate due to errors in the “excess energy” contribution. When the excess energy terms are kept as a constraint, the true potentials are recovered exactly.
Contaminant inclusion into protein crystals analyzed by electrospray mass spectrometry and X‐ray crystallography
Authors: Fontecilla‐Camps Juan Carlos , Halgand Frédéric , Forest Eric , Hirschler Joachim 
Published: July 2010

AbstractThe inclusion of protein contaminants into crystals of turkey egg white lysozyme (TEWL) was investigated by electrospray mass spectrometry of the dissolved crystals. The results show that significant amounts of the structurally related contaminant hen egg white lysozyme (HEWL) are included in the crystals of TEWL. The structurally unrelated contaminant RNAse A, on the other hand, is not included. The X‐ray diffraction data statistics of a hybrid TEWL/HEWL crystal and an uncontaminated TEWL crystal were of similar quality. This indicates that, even though the crystals contain much higher levels of the contaminant than one would have expected after a recrystallization experiment, they are still suitable for X‐ray diffraction experiments. However, attempts to detect the presence of the contaminant in the crystal by crystallographic structure refinement did not yield conclusive results.
Cloning, overexpression, purification, and spectroscopic characterization of human S100P
Authors: Gribenko Alexey , Lopez Maria M. , Richardson John M. , Makhatadze George I. 
Published: July 2010

AbstractThe calcium‐binding protein S100P has been found to be associated with human prostate cancer. We have overexpressed S100P in Escherichia coli using a T7 expression system. A rapid two‐step procedure for the isolation of overexpressed S100P leads to a preparation of >95% pure protein with a yield of ∼ 150 mg per liter of culture. The structural integrity of recombinant S100P was analyzed using CD and fluorescence spectroscopic techniques. The far‐UV CD shows that secondary structure of recombinant S100P consists predominantly of α‐helical structure. Both near‐UV CD and tyrosine fluorescence spectra show that aromatic residues are involved in the formation of a specific, well packed structure, indicating that the recombinant S100P protein adopts a compact folded conformation. Ca2+ has a profound effect on S100P structure. Near‐UV CD and fluorescence intensity of both internal (tyrosine) and external (ANS) probes suggest significant structural rearrangements in the tertiary structure of the molecule. The similarity of far‐UV CD spectrum of S100P in the presence and in the absence of Ca2+ suggests that Ca2+ binding has only minor effects on secondary structure.
Hirunorms are true hirudin mimetics. The crystal structure of human α‐thrombin‐hirunorm V complex
Authors: De Simone Giuseppina , Lombardi Angela , Galdiero Stefania , Nastri Flavia , Morte Rossella Della , Staiano Norma , Pedone Carlo , Bolognesi Martino , Pavone Vincenzo 
Published: July 2010

AbstractA novel class of synthetic, multisite‐directed thrombin inhibitors, known as hirunorms, has been described recently. These compounds were designed to mimic the binding mode of hirudin, and they have been proven to be very strong and selective thrombin inhibitors. Here we report the crystal structure of the complex formed by human α‐thrombin and hirunorm V, a 26‐residue polypeptide containing non‐natural amino acids, determined at 2.1 Å resolution and refined to anR‐factor of 0.176. The structure reveals that the inhibitor binding mode is distinctive of a true hirudin mimetic, and it highlights the molecular basis of the high inhibitory potency (Ki, is in the picomolar range) and the strong selectivity of hirunorm V.Hirunorm V interacts through the N‐terminal tetrapeptide with the thrombin active site in a nonsubstrate mode; at the same time, this inhibitor specifically binds through the C‐terminal segment to the fibrinogen recognition exosite. The backbone of the N‐terminal tetrapeptide Chg1″‐Val2″‐2‐Nal3″‐Thr4″(Chg, cyclohexyl‐glycine; 2‐Nal, β‐(2‐naphthyl)‐alanine) forms a short β‐strand parallel to thrombin main‐chain residues Ser2l4‐Gly219. The Chg1″side chain fills the S2 subsite, Val2″is located at the entrance of S1, whereas 2‐Nal3″side chain occupies the aryl‐binding site. Such backbone orientation is very close to that observed for the N‐terminal residues of hirudin, and it is similar to that of the synthetic retro‐binding peptide BMS‐183507, but it is opposite to the proposed binding mode of fibrinogen and of small synthetic substrates. Hirunorm V C‐terminal segment binds to the fibrinogen recognition exosite, similarly to what observed for hirudin C‐terminal tail and related compounds. The linker polypeptide segment connecting hirunorm V N‐and C‐terminal regions is not observable in the electron density maps. The crystallographic analysis proves the correctness of the design and it provides a compelling proof on the interaction mechanism for this novel class of high potency multisite‐directed synthetic thrombin inhibitors.
Exploring sequence constraints on an interhelical turn using in vivo selection for catalytic activity
Authors: Macbeath Gavin , Kast Peter , Hilvert Donald 
Published: July 2010

AbstractThe role of interhelical turns in determining protein structure has been investigated previously in relatively simple four‐helix‐bundle proteins using combinatorial mutagenesis coupled with screening for functional variants. To assess the tolerance to sequence substitution of a short, interhelical turn in a larger, more complicated protein, we have exploited a more sensitive in vivo selection for catalytic activity. Randomization of three solvent‐exposed turn residues in Escherichia coli chorismate mutase (Ala65, His66, and His67), followed by selection, indicated that > 63% of tripeptides, including some with significantly altered backbone conformations, can functionally replace the native sequence. The increased sensitivity of the catalytic assay allowed optimal sequences to be distinguished from less appropriate ones, revealing a statistically significant preference for hydrophilic residues in solvent‐exposed positions. It also enabled investigation of the extent to which either secondary structure or tertiary interactions influence substitution patterns. Randomization of an α‐helical residue (Lys64), together with the adjacent solvent‐exposed tripeptide, Ala65‐His66‐His67, showed that the secondary structure at position 64 does not limit the range of side chains allowed at this site. In contrast, randomization of a buried turn residue (Leu68), together with the same tripeptide, revealed an extremely strict requirement for hydrophobic aliphatic amino acids at this position. The strong constraint imposed by the tertiary interaction, in contrast to the weak influence of secondary structure, has important implications for protein design.
Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin‐induced conformers of antithrombin
Authors: Rezaie Alireza R. 
Published: July 2010

AbstractA pentasaccharide (PS) fragment of heparin capable of activating antithrombin (AT) markedly accelerates the inhibition of factor Xa by AT, but has insignificant effect on inhibition of thrombin. For inhibition of thrombin, the bridging function of a longer polysaccharide chain is required to accelerate the reaction. To study the basis for the similar reactivity of thrombin with the native or heparin‐activated conformers of AT, several residues surrounding the active site pocket of thrombin were targeted for mutagenesis study. Leu99 and Glu192, the variant residues influencing the S2 and S3 subsite specificity of thrombin were replaced with Tyr and Gin. The Tyr60a, Pro60b, Pro60c, and Trp60d residues forming part of the S2 specificity pocket were deleted from the B‐insertion loop of the wild‐type and Leu99/Glu192 Tyr/Gln thrombins. Kinetic studies indicated that the reactivities of all mutants with AT were moderately or severely impaired. Although heparin largely corrected the defect in reactivities, it also markedly elevated the stoichiometrics of inhibition with the mutants. Interestingly, PS also accelerated AT inhibition of the mutants 5‐68‐fold, suggesting that the mutants are able to discriminate between the native and activated conformers of AT. Based on these results and the recent crystal structure determination of AT in complex with PS, a model for thrombin‐AT interaction is proposed in which the S2 and S3 subsite residues of thrombin are critical for recognition of the P2 and P3 residues of AT in the native conformation. In the activated conformation, other residues are made accessible for interaction with the protease, and the similar reactivity of thrombin with the native and heparin‐activated conformers of AT may be coincidental. The results further suggest that the S2 and S3 subsite residues are crucial in controlling the partitioning of the thrombin‐AT intermediate into the alternative inhibitory or substrate pathways of the reaction.