bluuebunny/crossref_metadata_2025 huggingface dataset:
Ion‐specific modulation of protein interactions: Anion‐induced, reversible oligomerization of a fusion protein
Authors: Gokarn Yatin R. , Fesinmeyer R. Matthew , Saluja Atul , Cao Shawn , Dankberg Jane , Goetze Andrew , Jr. Richard L. Remmele , Narhi Linda O. , Brems David N.
Published: February 2009

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt‐dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion‐specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1‐Fc with two, C‐terminal peptide arms linked via penta‐glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self‐association upon salt addition. The sedimentation coefficient (sw) and hydrodynamic diameter (DH) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in sw and DH, reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The DH and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z‐average molecular weight (Mz) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self‐association for an IgG1‐Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1‐Fc and PbA, indicative of PbA self‐assembly being mediated through its peptide arms. Self‐association increased with pH, NaCl concentration, and anion size (I− > Br− > Cl− > F−) but could be inhibited using soluble Trp‐, Phe‐, and Leu‐amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self‐association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self‐association occurs via aromatic and hydrophobic interactions between the ..xx..xxx..xx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.
High tolerance to simultaneous active‐site mutations in TEM‐1 β‐lactamase: Distinct mutational paths provide more generalized β‐lactam recognition
Authors: De Wals Pierre‐Yves , Doucet Nicolas , Pelletier Joelle N.
Published: February 2009

The diversity in substrate recognition spectra exhibited by various β‐lactamases can result from one or a few mutations in the active‐site area. Using Escherichia coli TEM‐1 β‐lactamase as a template that efficiently hydrolyses penicillins, we performed site‐saturation mutagenesis simultaneously on two opposite faces of the active‐site cavity. Residues 104 and 105 as well as 238, 240, and 244 were targeted to verify their combinatorial effects on substrate specificity and enzyme activity and to probe for cooperativity between these residues. Selection for hydrolysis of an extended‐spectrum cephalosporin, cefotaxime (CTX), led to the identification of a variety of novel mutational combinations. In vivo survival assays and in vitro characterization demonstrated a general tendency toward increased CTX and decreased penicillin resistance. Although selection was undertaken with CTX, productive binding (KM) was improved for all substrates tested, including benzylpenicillin for which catalytic turnover (kcat) was reduced. This indicates broadened substrate specificity, resulting in more generalized (or less specialized) variants. In most variants, the G238S mutation largely accounted for the observed properties, with additional mutations acting in an additive fashion to enhance these properties. However, the most efficient variant did not harbor the mutation G238S but combined two neighboring mutations that acted synergistically, also providing a catalytic generalization. Our exploration of concurrent mutations illustrates the high tolerance of the TEM‐1 active site to multiple simultaneous mutations and reveals two distinct mutational paths to substrate spectrum diversification.
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

Although 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

Proteins 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

Although 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

Thermal 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

We 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

The 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

An 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

We 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

BM 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

Recent 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

The 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

A 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

Based 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

The 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

The 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.
Macromolecular crystal growth experiments on international microgravity laboratory – 1
Authors: Day John , McPherson Alexander
Published: February 2009

Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory — 1 (IML‐1), flown January 22–29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion‐based device (PCG) and a liquid‐liquid diffusion‐based instrument (CRYOSTAT). The experiments in each device were run at 20–22 °C and at colder temperatures.

Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid‐liquid diffusion were more than 10‐fold greater in total volume than any STMV crystals previously grown in the laboratory.

X‐ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 Å resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 Å resolution.

Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes.
Differences in the amino acid distributions of 310‐helices and α‐helices
Authors: Karpen Mary E. , De Haseth Pieter L. , Neet Kenneth E.
Published: February 2009

Local determinants of 310‐helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5‐length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 310‐helices and multiple‐turn conformations, had strong amino acid sequence patterns not evident among α‐helices. Aspartate occurred over twice as frequently in the N‐cap position of 310‐helices as in the N‐cap position of α‐helices. Unlike α‐helices, 310‐helices had few C‐termini ending in a left‐handed α conformation; most 310 C‐caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 310‐ and α‐helices were also apparent, producing amphipathic 310‐helices. Local interactions that stabilize 310‐helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of α‐helices have been postulated to proceed through reverse‐turn and 310‐helix intermediates, sequence differences between 310‐ and α‐helices can also lend insight into factors influencing α‐helix initiation and propagation.
Purification and properties of the cellular prion protein from Syrian hamster brain
Authors: Pan Keh‐Ming , Stahl Neil , Prusiner Stanley B.
Published: February 2009

The cellular prion protein (PrPC) is encoded by a chromosomal gene, and its scrapie isoform (PrPSc) features in all aspects of the prion diseases. Prior to the studies reported here, purification of PrPC has only been accomplished using immunoaffinity chromatography yielding small amounts of protein. Brain homogenates contain two PrPC forms designated PrPC‐I and ‐II. These proteins were purified from a microsomal fraction by detergent extraction and separated by immobilized Cu2+ ion affinity chromatography. PrPC‐II appears to be generated from PrPC‐I by limited proteolysis of the N‐terminus. Fractions enriched for PrPC‐I were purified further by cation‐exchange chromatography and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Greater than 90% of the final product migrated as a broad band of Mr 33–35 kDa as judged by silver staining after SDS‐PAGE. Digestion of PrPC‐I with peptide‐N‐glycosidase (PNGase) compressed the band and shifted its mobility giving an Mr of 27 kDa. The protocol described should be amenable to large‐scale preparation of PrPC, enabling physical comparisons of PrPC and PrPSc.
Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two‐dimensional NMR spectroscopy
Authors: Wittekind Michael , Rajagopal Ponni , Branchini Bruce R. , Reizer Jonathan , Saier Milton H. , Klevit Rachel E.
Published: February 2009

The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two‐dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse‐detected 2D (1H‐15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann‐Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross‐peaks that are not resolved in 2D homonuclear 1H spectra. Distance constraints derived from NOESY cross‐peaks, hydrogen‐bonding patterns derived from 1H–2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X‐PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 Å (N, Cα, C', O atoms), excluding the segments containing residues 45–59 and 84–88. The structure is comprised of a four‐stranded antiparallel β‐sheet with two antiparallel α‐helices on one side of the sheet. The active‐site His 15 residue serves as the N‐cap of α‐helix A, with its Nδ1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. The existence of a hydrogen bond between the side‐chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the β‐turn comprised of residues 37–40. If the βαββαβ(α) folding opology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of βαββαβ proteins that include acylphosphatase and the RNA‐binding domains of the U1 snRNP A and hnRNP C proteins.
Modeling the antigen combining site of an anti‐dinitrophenyl antibody, ANO2
Authors: Bassolino‐Klimas Donna , Bruccoleri Robert E. , Subramaniam Shankar
Published: February 2009

A model structure has been constructed for a monoclonal anti‐dinitrophenyl antibody. The antibody, ANO2, has been sequenced and cloned (Anglister, J., Frey, T., & McConnell, H.M., 1984, Biochemistry 23, 1138–1142). Its amino acid sequence shows striking homology with the anti‐lysozyme Fab fragments HyHel5 (83%) and HyHel10 (73%). Based on this homology, a model for the ANO2 variable heavy and variable light chain framework was constructed using a hybrid of the HyHel5 light chain and the HyHel10 heavy chain backbone, omitting the hypervariable loops. These coordinates were used as scaffolds for the model building of ANO2.

The CONGEN conformational sampling algorithm (Bruccoleri, R.E. & Karplus, M., 1987, Biopolymers 26, 127–196) was used to model the six hypervariable loops that contain the antigen‐combining site. All the possible conformations of the loop backbones were constructed and the best loop structures were selected using a combination of the CHARMM potential energy function and evaluation of the solvent‐accessible surface area of the conformers. The order in which the loops were searched was carried out based on the relative locations of the loops with reference to the framework of the β‐barrel, namely, L2‐H1‐L3‐H2‐H3‐L1. The model structures thus obtained were compared to the high resolution X‐ray structure (Brünger, A.T., Leahy, D.J., Hynes, T.R., & Fox, R.O., 1991, J. Mol. Biol. 221, 239–256).
A detailed consideration of a principal domain of vertebrate fibrinogen and its relatives
Authors: Doolittle Russell F.
Published: February 2009

Vertebrate fibrinogen is a complex multidomained protein, the structure of which has been inferred mainly from electron microscopy and amino acid sequence studies. Among its most prominent features are two terminal globules, moieties that are mostly composed of the carboxyl‐terminal two‐thirds of the β and γ chains. Sequences homologous to the latter segments are found in several other animal proteins, always as the carboxyl‐terminal contributions. An alignment of 15 amino acid sequences from various fibrinogens and related proteins has been used to make judgments about secondary structure. The nature of amino acids at each position in the alignment was used to distinguish α helices and β structure on the one hand from loops and turns on the other, and the resulting assignments compared with predictions of secondary structure by other methods. Additionally, constraints imposed by the locations of cystines, carbohydrate attachment residues, and proteinase‐sensitive points provided further insights into the general organization of the postulated secondary structures. Other ancillary data, including the effects of bound calcium and the locations of labeled or variant residues, were also considered. An intriguing similarity to a portion of the recently reported structure of a calcium‐dependent lectin is noted.
A database of protein structure families with common folding motifs
Authors: Holm Liisa , Ouzounis Christos , Sander Chris , Tuparev Georg , Vriend Gert
Published: February 2009

The availability of fast and robust algorithms for protein structure comparison provides an opportunity to produce a database of three‐dimensional comparisons, called families of structurally similar proteins (FSSP). The database currently contains an extended structural family for each of 154 representative (below 30% sequence identity) protein chains. Each data set contains: the search structure; all its relatives with 70–30% sequence identity, aligned structurally; and all other proteins from the representative set that contain substructures significantly similar to the search structure. Very close relatives (above 70% sequence identity) rarely have significant structural differences and are excluded. The alignments of remote relatives are the result of pairwise all‐against‐all structural comparisons in the set of 154 representative protein chains. The comparisons were carried out with each of three novel automatic algorithms that cover different aspects of protein structure similarity. The user of the database has the choice between strict rigid‐body comparisons and comparisons that take into account interdomain motion or geometrical distortions; and, between comparisons that require strictly sequential ordering of segments and comparisons, which allow altered topology of loop connections or chain reversals. The data sets report the structurally equivalent residues in the form of a multiple alignment and as a list of matching fragments to facilitate inspection by three‐dimensional graphics. If substructures are ignored, the result is a database of structure alignments of full‐length proteins, including those in the twilight zone of sequence similarity. The database makes explicitly visible architectural similarities in the known part of the universe of protein folds and may be useful for understanding protein folding and for extracting structural modules for protein design. The data sets are available via Internet.
Dominant role of local dipoles in stabilizing uncompensated charges on a sulfate sequestered in a periplasmic active transport protein
Authors: He Jing J. , Quiocho Florante A.
Published: February 2009

Electrostatic interactions are among the key factors determining the structure and function of proteins. Here we report experimental results that illuminate the functional importance of local dipoles to these interactions. The refined 1.7‐Å X‐ray structure of the liganded form of the sulfate‐binding protein, a primary sulfate active transport receptor of Salmonella typhimurium, shows that the sulfate dianion is completely buried and bound by hydrogen bonds (mostly main‐chain peptide NH groups) and van der Waals forces. The sulfate is also closely linked, via one of these peptide units, to a His residue. It is also adjacent to the N‐termini of three α‐helices, of which the two shortest have their C‐termini “capped” by Arg residues. Site‐directed mutagenesis of the recombinant Escherichia coli sulfate receptor had no effect on sulfate‐binding activity when an Asn residue was substituted for the positively charged His and the two Arg (changed singly and together) residues. These results, combined with other observations, further solidify the idea that stabilization of uncompensated charges in a protein is a highly localized process that involves a collection of local dipoles, including those of peptide units confined to the first turns of helices. The contribution of helix macrodipoles appears insignificant.
Identification of cysteine ligands in metalloproteins using optical and NMR spectroscopy: Cadmium‐substituted rubredoxin as a model [Cd(CysS)4]2‐ center
Authors: Henehan Colin J. , Pountney Dean L. , Vašák Milan , Zerbe Oliver
Published: February 2009

Optical and NMR methods are presented for the identification of cysteine ligands in Cd‐substituted metalloproteins, in particular those containing zinc‐fingerlike motifs, using Cd‐substituted Desulfovibrio gigas rubredoxin (Cd‐Rd) as a model [Cd(CysS)4]2‐ complex. The 113Cd NMR spectrum of Cd‐Rd contains a single 113Cd resonance with a chemical shift position (723.6 ppm) consistent with tetrathiolate metal coordination. The proton chemical shifts of the four cysteine ligands were obtained from one‐dimensional heteronuclear (1H‐113Cd) multiple quantum coherence (HMQC) and total coherence spectroscopy (TOCSY)‐relayed HMQC experiments. In addition, sequential assignments were made for two short cysteine‐containing stretches of the polypeptide chain using a combination of homonuclear proton correlated spectroscopy, TOCSY, and nuclear Overhauser effect spectroscopy experiments, enabling sequence‐specific heteronuclear 3J(1Hβ‐113Cd) coupling constants for each cysteine to be determined. The magnitude of these couplings (0–38 Hz) follows a Karplus‐like dependence with respect to the H
β
‐C
β
‐S
γ
‐Cd dihedral angles, inferred from the crystal structure of the native protein. The difference absorption envelope (Cd‐Rd vs. apo‐Rd) reveals three distinct transitions with Gaussian‐resolved maxima located at 213, 229, and 245 nm, which are paralleled by dichroic features in the corresponding difference CD and magnetic CD spectra. Based on the optical electronegativity theory of Jørgensen, the lowest energy transition has been attributed to a CysS‐Cd(II) charge‐transfer excitation (245, 26,000 M−1 cm−1) with a molar extinction coefficient per cysteine of 6,500 M−1 cm−1. It is proposed that the intensity of this band can be used as a sensitive measure of the number of cysteine ligands present in Cd(CysS)4‐n
X
n
centers.
Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant
Authors: Merlino Antonello , Avella Giovanna , Di Gaetano Sonia , Arciello Angela , Piccoli Renata , Mazzarella Lelio , Sica Filomena
Published: February 2009

A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, which is critically dependent on their ability to reach the cytosol and to evade the action of the ribonuclease inhibitor (RI). The cytotoxicity and antitumor activity of bovine seminal ribonuclease (BSRNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, are peculiar properties of the swapped form. A dimeric variant (HHP2‐RNase) of human pancreatic RNase, in which the enzyme has been engineered to reproduce the sequence of BSRNase helix‐II (Gln28→Leu, Arg31→Cys, Arg32→Cys, and Asn34→Lys) and to eliminate a negative charge on the surface (Glu111→Gly), is also extremely cytotoxic. Surprisingly, this activity is associated also to the unswapped form of the protein. The crystal structure reveals that on this molecule the hinge regions, which are highly disordered in the unswapped form of BSRNase, adopt a very well‐defined conformation in both subunits. The results suggest that the two hinge peptides and the two Leu28 side chains may provide an anchorage to a transient noncovalent dimer, which maintains Cys31 and Cys32 of the two subunits in proximity, thus stabilizing a quaternary structure, similar to that found for the noncovalent swapped dimer of BSRNase, that allows the molecule to escape RI and/or to enhance the formation of the interchain disulfides.
Morphological, electrical and optical properties of sputtered Mo thin films on flexible substrates
Authors: Bollero A. , Andrés M. , García C. , Abajo J. de , Gutiérrez M. T.
Published: February 2009

Molybdenum (Mo) films were deposited onto Kapton® substrates with different thickness by dc‐magnetron sputtering in order to be used as back contact layer in flexible solar cells. The morphology, electrical resistance and reflectance of the films have been studied as a function of the deposition parameters and the thickness of the polymer films. The lowest resistivity and highest reflectance have been obtained for those films deposited at a reduced working gas pressure of 0.6 Pa. The evolution of these properties is shown to be strongly dependent on the microstructure of the films. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Electrodeposition behavior of noble metals in ordered macroporous silicon
Authors: Matsumoto Tasuku , Kobayashi Katsutoshi , Fukami Kazuhiro , Sakka Tetsuo , Ogata Yukio H.
Published: February 2009

Electrodeposition of noble metals (Pt, Pd, and Au) in ordered macropores prepared in p‐type Si under dark condition was investigated. Pt deposition proceeded continuously from the pore bottoms, and ordered micro‐rods were obtained. Pd deposition proceeded on the whole pore walls, and discontinuous micro‐tubes were obtained. Au deposition proceeded at 10‐20 μm depth from the pore openings where a high immersion plating rate was observed without applied potential, and short micro‐rods were obtained. At the pore bottoms a small amount of deposition was observed. The different behaviors of electrodeposition depending on the metals were compared and discussed. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
EPR and photoluminescence diagnostics of singlet oxygen generation on porous silicon surface
Authors: Konstantinova E. A. , Demin V. A. , Ryabchikov Yu. V. , Gongalskiy M. B. , Kashkarov P. K.
Published: February 2009

Electron paramagnetic resonance and photoluminescence spectroscopy are used to investigate photosensitized generation of singlet oxygen in the porous silicon layers. The singlet oxygen concentration in the samples was estimated at various oxygen pressures. The time of energy transfer from excitons confined in Si nanocrystals to adsorbed O2 molecules on silicon nanocrystal surface and photosensitization efficiency are found to depend on the porosity of the samples. The singlet oxygen generation efficiency increases strongly for porous silicon with high (>80%) porosity. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)