bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational changes within the HLA‐A1:MAGE‐A1 complex induced by binding of a recombinant antibody fragment with TCR‐like specificity
Authors: Kumar Pravin , Vahedi‐Faridi Ardeschir , Saenger Wolfram , Ziegler Andreas , Uchanska‐Ziegler Barbara 
Published: February 2009

AbstractAlthough there is X‐ray crystallographic evidence that the interaction between major histocompatibility complex (MHC, in humans HLA) class I molecules and T cell receptors (TCR) or killer cell Ig‐like receptors (KIR) may be accompanied by considerable changes in the conformation of selected residues or even entire loops within TCR or KIR, conformational changes between receptor‐bound and ‐unbound MHC class I molecules of comparable magnitude have not been observed so far. We have previously determined the structure of the MHC class I molecule HLA‐A1 bound to a melanoma antigen‐encoding gene (MAGE)‐A1‐derived peptide in complex with a recombinant antibody fragment with TCR‐like specificity, Fab‐Hyb3. Here, we compare the X‐ray structure of HLA‐A1:MAGE‐A1 with that complexed with Fab‐Hyb3 to gain insight into structural changes of the MHC molecule that might be induced by the interaction with the antibody fragment. Apart from the expulsion of several water molecules from the interface, Fab‐Hyb3 binding results in major rearrangements (up to 5.5 Å) of heavy chain residues Arg65, Gln72, Arg145, and Lys146. Residue 65 is frequently and residues 72 and 146 are occasionally involved in TCR binding‐induced conformational changes, as revealed by a comparison with MHC class I structures in TCR‐liganded and ‐unliganded forms. On the other hand, residue 145 is subject to a reorientation following engagement of HLA‐Cw4 and KIR2DL1. Therefore, conformational changes within the HLA‐A1:MAGE‐A1:Fab‐Hyb3 complex include MHC residues that are also involved in reorientations in complexes with natural ligands, pointing to their central importance for the peptide‐dependent recognition of MHC molecules.
Knitting and untying the protein network: Modulation of protein ensembles as a therapeutic strategy
Authors: Gordo Susana , Giralt Ernest 
Published: February 2009

AbstractProteins constitute the working machinery and structural support of all organisms. In performing a given function, they must adopt highly specific structures that can change with their level of activity, often through the direct or indirect action of other proteins. Indeed, proteins typically function within an ensemble, rather than individually. Hence, they must be sufficiently flexible to interact with each other and execute diverse tasks. The discovery that errors within these groups can ultimately cause disease has led to a paradigm shift in drug discovery, from an emphasis on single protein targets to a holistic approach whereby entire ensembles are targeted.
Amino acid binding by the Class I aminoacyl‐tRNA synthetases: Role for a conserved proline in the signature sequence
Authors: Burbaum Jonathan J. , Schimmel Paul 
Published: February 2009

AbstractAlthough partial or complete three‐dimensional structures are known for three Class I aminoacyl‐tRNA synthetases, the amino acid‐binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl‐tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl‐tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.‐P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449–457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300‐fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the “signature sequence” of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl‐tRNA synthetases and, in the known structures, it is in the nucleotide‐binding fold as part of a loop between the end of a β‐strand and the start of an α‐helix. The phenotype of the mutant and the stability and affinity for methionine of the wild‐type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C‐terminus of the signature sequence. (The importance of this position in amino acid binding was demonstrated from studies on another closely related Class I synthetase [Clarke, N.D., Lien, D.C., & Schimmel, P., 1988, Science 240, 521–523].) Because the target proline is conserved in Class I enzymes, it is not likely to determine amino acid side‐chain specificity directly. Instead, structural modeling suggests that this proline acts as a wedge to open an amino acid‐binding cleft between adjacent strands of the β‐sheet.
Reversible unfolding and refolding behavior of a monomeric aldolase from staphylococcus aureus
Authors: Rudolph Rainer , Siebendritt Renate , Kiefhaber Thomas 
Published: February 2009

AbstractThermal and GdmCl‐induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a ΔG0 value of ‐9 ± 2 kJ/mol at 20 °C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 °C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of α/β barrel proteins.
Identification of the adipocyte acid phosphatase as a PAO‐sensitive tyrosyl phosphatase
Authors: Shekels Laurie L. , Smith Anne J. , Bernlohr David A. , Van Etten Robert L. 
Published: February 2009

AbstractWe have partially purified an 18‐kDa cytoplasmic protein from 3T3‐L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 μM), and the inactivation could be reversed by the dithiol, 2,3‐dimercaptopropanol (Kreact = 23 μM), but not the monothiol, 2‐mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAPα and HAAPβ (human adipocyte acid phosphatase), which are distinguished by a 34‐amino acid isoform‐specific domain. Sequence analysis shows HAAPα and HAAPβ share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAPβ has been cloned into Escherichia coli, expressed, and purified as a glutathione S‐transferase fusion protein. Recombinant HAAPβ was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 μM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.
The disulfide bond pairing of the pheromones Er‐1 and Er‐2 of the ciliated protozoan Euplotes raikovi
Authors: Stewart Albert E. , Raffioni Simona , Bradshaw Ralph A. , Chaudhary Tanuja , Chait Brian T. , Luporini Pierangelo 
Published: February 2009

AbstractThe disulfide pairings of the two Euplotes raikovi pheromones Er‐1 and Er‐2 have been determined by chemical and mass spectrometric analyses. Cystine‐linked peptides from thermolytic digestions of the native molecules were purified by reverse‐phase high performance liquid chromatography and identified in the known sequences to make the assignments. The same pairing, Cys(I)–Cys(IV), Cys(II)–Cys(VI), and Cys(III)–Cys(V), was found in both pheromones, suggesting that this pattern occurs commonly throughout this family of molecules. This arrangement of disulfides indicates that the three‐dimensional structure is defined by three loops, which can vary in size and charge distribution from one pheromone to another.
Complete enzymatic deglycosylation of native sex steroid‐binding protein (SBP or SHBG) of human and rabbit plasma: Effect on the steroid‐binding activity
Authors: Petra Philip H. , Zhang Wei , Griffin Patrick R. , Yates John R. , Moore Katherine 
Published: February 2009

AbstractAn enzymatic procedure for the complete removal of the N‐linked and O‐linked oligosaccharide side chains of the sex steroid‐binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N‐glycanase, neuraminidase, and O‐glycanase and was monitored by SDS‐PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N‐glycanase generated a major 39,777‐Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar‐free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066–19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino‐terminus. The N‐ and O‐linked side chains of human SBP were removed by sequential digestion with N‐glycanase and neuraminidase/O‐glycanase. A 38,771‐Da protein was generated, which agrees well with the molecular weight of the sugar‐free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584–7590). N‐deglycosylation of human and rabbit SBP has no effect on the steroid‐binding activity, but removal of the O‐linked side chains of N‐deglycosylated human SBP results in an apparent 50% loss of steroid‐binding activity and an increase in the Kd for the binding of 5α‐dihydrotestosterone from 0.3 nM to 0.9 nM. There are no changes in steroid‐binding specificity. The apparent loss of activity of O‐deglycosylated human SBP is probably due to the small changes in the Kd, which could influence the equilibrium concentration of bound SBP when measured under standard assay conditions. We conclude that deglycosylation has very little effect on steroid‐binding activity and that the oligosaccharide side chains must serve other functions in the physiology of SBP.
Role of interchain α‐helical hydrophobic interactions in Ca2+ affinity, formation, and stability of a two‐site domain in troponin C
Authors: Monera Oscar D. , Shaw Gary S. , Sykes Brian D. , Kay Cyril M. , Hodges Robert S. , Zhu Bing‐Yan 
Published: February 2009

AbstractWe have previously shown that a 34‐residue synthetic peptide representing the calcium‐binding site III of troponin C formed a symmetric two‐site dimer consisting of two helix‐loop‐helix motifs arranged in a head‐to‐tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280–283). In this study the hydrophobicities of the α‐helices were altered by replacing L‐98 and F‐102 in the N‐terminal region and/or I‐121 and L‐122 in the C‐terminal region with alanine residues. Our results showed that substitution of hydrophobic residues either in the N‐ or C‐terminal region have little effect on α‐helix formation but resulted in a 100‐ and 300‐fold decrease in Ca2+ affinity, respectively. Simultaneous substitution of both hydrophobes in the N‐ and C‐terminal region resulted in a 1,000‐fold decrease in Ca2+ affinity. Data from guanidine hydrochloride denaturation studies suggested that intermolecular interactions occur and that the less hydrophobic analogs had a lower overall conformational stability. These data support the contention that the hydrophobic residues are important in the formation of the two‐site domain in troponin C, and this hydrophobic association stabilizes Ca2+ affinity.
Mapping of the catalytic site of CHO‐t‐PA and the t‐PA variant BM 06.022 by synthetic inhibitors and substrates
Authors: Stürzebecher J. , Neumann U. , Kohnert U. , Kresse G.‐B. , Fischer S. 
Published: February 2009

AbstractBM 06.022 is a t‐PA deletion variant that is produced as inactive inclusion bodies inEscherichia coliand transformed into the native form by an in vitro refolding process. Until now, no X‐ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine‐derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t‐PA. Our data reveal that the single‐chain as well as the two‐chain form of BM 06.022 and native t‐PA are similar in catalytic and in inhibitor binding properties. This indicates that the active site and the highly complex rearrangement of t‐PA upon cleavage of the Arg275‐Ile276bond are maintained in BM 06.022.
The accessibility of etheno‐nucleotides to collisional quenchers and the nucleotide cleft in G‐ and F‐actin
Authors: Root Douglas D. , Reisler Emil 
Published: February 2009

AbstractRecent publication of the atomic structure of G‐actin (Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes, K.C., 1990, Nature 347, 37–44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno‐nucleotides bound to G‐ and F‐actin were examined in order to assess polymerization‐related changes in the nucleotide phosphate site. The Mg2+‐induced polymerization of actin quenched the fluorescence of the etheno‐nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F‐actin than G‐actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G‐ and F‐actin was greatest for I−, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization‐related movements of charged residues in the atomic structure of G‐actin are suggested. The binding of S‐1 to ϵ‐ATP‐G‐actin increased the accessibility of the etheno group to I− even over that in Mg2+‐polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S‐1 to actin. Thus, the binding of S‐1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin. The pH dependence of collisional quenching shows that the cleft region is more accessible to collisional quenchers at pH 7 than at higher pH and suggests that changes in the environment of the cleft might contribute to the faster polymerization rates observed at lower pH.
Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins
Authors: Park Kyusung , Perczel András , Fasman Gerald D. 
Published: February 2009

AbstractThe interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X‐ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G.D., 1991, Protein Eng. 4, 669–679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived “pure” CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five‐component spectra so obtained from the deconvolution consisted of two different types of α helices (the α helix in the soluble domain and the αT helix, for the transmembrane α helix), a β‐pleated sheet, a class C‐like spectrum related to β turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the αT helix was characterized by a positive red‐shifted band in the range 195–200 nm (+95,000 deg cm2 dmol−1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222‐nm band (–50,000 and −60,000 deg cm2 dmol−1, respectively) in comparison with the regular α helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, ‐30,000, and ‐30,000 deg cm2 dmol−1, respectively.
Sequence analysis of peptide mixtures by automated integration of Edman and mass spectrometric data
Authors: Johnson R. S. , Walsh K. A. 
Published: February 2009

AbstractA computer algorithm is described that utilizes both Edman and mass spectrometric data for simultaneous determination of the amino acid sequences of several peptides in a mixture. Gas phase sequencing of a peptide mixture results in a list of observed amino acids for each cycle of Edman degradation, which by itself may not be informative and typically requires reanalysis following additional chromatographic steps. Tandem mass spectrometry, on the other hand, has a proven ability to analyze sequences of peptides present in mixtures. However, mass spectrometric data may lack a complete set of sequence‐defining fragment ions, so that more than one possible sequence may account for the observed fragment ions. A combination of the two types of data reduces the ambiguity inherent in each. The algorithm first utilizes the Edman data to determine all hypothetical sequences with a calculated mass equal to the observed mass of one of the peptides present in the mixture. These sequences are then assigned figures of merit according to how well each of them accounts for the fragment ions in the tandem mass spectrum of that peptide. The program was tested on tryptic and chymotryptic peptides from hen lysozyme, and the results are compared with those of another computer program that uses only mass spectral data for peptide sequencing. In order to assess the utility of this method the program is tested using simulated mixtures of varying complexity and tandem mass spectra of varying quality.
Active site mutants of human cyclophilin A separate peptidyl‐prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition
Authors: Zydowsky Lynne D. , Etzkorn Felicia A. , Chang Howard Y. , Ferguson Stephen B. , Stolz Lesley A. , Ho Susanna I. , Walsh Christopher T. 
Published: February 2009

AbstractBased on recent X‐ray structural information, six site‐directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site‐H54, R55, F60, Q111, F113, and H126–have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.‐M., & Walsh, C.T., 1991a, Biochemistry 30, 2306–2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis‐trans peptidyl‐prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3–15% of the catalytic efficiency (kcat/Km) of wild‐type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild‐type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.
Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols
Authors: Bhat Rajiv , Timasheff Serge N. 
Published: February 2009

AbstractThe preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and β‐lactoglobulin with polyethylene glycols (PEGs) of molecular weight 200–6,000 have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein. The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by Rg = aM0.55. The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24, 6756–6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.
Molecular dynamics studies of a DNA‐binding protein: 1. A comparison of the trp repressor and trp aporepressor aqueous simulations
Authors: Howard Allison E. , Kollman Peter A. 
Published: February 2009

AbstractThe results of two 30‐ps molecular dynamics simulations of the trp repressor and trp aporepressor proteins are presented in this paper. The simulations were obtained using the AMBER molecular mechanical force field and in both simulations a 6‐Å shell of TIP3P waters surrounded the proteins. The trp repressor protein is a DNA‐binding regulatory protein and it utilizes a helix‐turn‐helix (D helix‐turn‐E helix) motif to interact with DNA. The trp aporepressor, lacking two molecules of the l‐tryptophan corepressor, cannot bind specifically to DNA. Our simulations show that the N‐ and C‐termini and the residues in and near the helix‐turn‐;helix motifs are the most mobile regions of the proteins, in agreement with the X‐ray crystallographic studies. Our simulations also find increased mobility of the residues in the turn‐D helix‐turn regions of the proteins. We find the average distance separating the DNA‐binding motifs to be larger in the repressor as compared to the aporepressor. In addition to examining the protein residue fluctuations and deviations with respect to X‐ray structures, we have also focused on backbone dihedral angles and corepressor hydrogen‐bonding patterns in this paper.