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Hydrophobic Residues (hydrophobic + residue)
Selected AbstractsThermosynechoccus elongatus DpsA binds Zn(II) at a unique three histidine-containing ferroxidase center and utilizes O2 as iron oxidant with very high efficiency, unlike the typical Dps proteinsFEBS JOURNAL, Issue 4 2010Flaminia Alaleona The cyanobacterium Thermosynechococcus elongatus is one the few bacteria to possess two Dps proteins, DpsA-Te and Dps-Te. The present characterization of DpsA-Te reveals unusual structural and functional features that differentiate it from Dps-Te and the other known Dps proteins. Notably, two Zn(II) are bound at the ferroxidase center, owing to the unique substitution of a metal ligand at the A-site (His78 in place of the canonical aspartate) and to the presence of a histidine (His164) in place of a hydrophobic residue at a metal-coordinating distance in the B-site. Only the latter Zn(II) is displaced by incoming iron, such that Zn(II),Fe(III) complexes are formed upon oxidation, as indicated by absorbance and atomic emission spectroscopy data. In contrast to the typical behavior of Dps proteins, where Fe(II) oxidation by H2O2 is about 100-fold faster than by O2, in DpsA-Te the ferroxidation efficiency of O2 is very high and resembles that of H2O2. Oxygraphic experiments show that two Fe(II) are required to reduce O2, and that H2O2 is not released into solution at the end of the reaction. On this basis, a reaction mechanism is proposed that also takes into account the formation of Zn(II),Fe(III) complexes. The physiological significance of the DpsA-Te behavior is discussed in the framework of a possible localization of the protein at the thylakoid membranes, where photosynthesis takes place, with the consequent increased formation of reactive oxygen species. Structured digital abstract ,,MINT-7312099: DpsA (uniprotkb:Q8DL82) and DpsA (uniprotkb:Q8DL82) bind (MI:0407) by x-ray crystallography (MI:0114) [source] Biochemical characterization of human cathepsin X revealed that the enzyme is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidaseFEBS JOURNAL, Issue 17 2000Ivica Klemen Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of ,,33 kDa and pI 5.1,5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (Ki = 1.7,15.0 nm), but poorly or not at all by stefin B (Ki > 250 nm) and l -kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA-074 and GFG-semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1, position, although the enzyme cleaved all P1, residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide-blocked C-terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o -aminobenzoic acid-peptidyl- N -[2,-dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (kcat/Km,,5.0 × 103 m,1·s,1) were degraded ,,25-fold less efficiently than the carboxypeptidase substrates (kcat/Km , 120.0 × 103 m,1·s,1). [source] The importance of valine 114 in ligand binding in ,2 -adrenergic receptorPROTEIN SCIENCE, Issue 1 2010Makoto Arakawa Abstract G-protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. ,2 -Adrenergic receptor (,2 -AR) is a well-studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand-binding region of ,2 -AR using the recently solved high-resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure-function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster ,2 -AR by site-directed mutagenesis. We replaced V114 in hamster ,2 -AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G-protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side-chains but also the aryl-ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in ,2 -AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding. [source] X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3,Å resolution using a twinned crystalACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Kimihiko Mizutani Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P63. Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33,2.3,Å. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD. [source] Subsite specificity of trypanosomal cathepsin L-like cysteine proteasesFEBS JOURNAL, Issue 9 2001Probing the S2 pocket with phenylalanine-derived amino acids The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards d/l -Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mm,1·s,1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs. [source] A Class of Cationic Triblock Amphiphilic Oligopeptides as Efficient Gene-Delivery Vectors,ADVANCED MATERIALS, Issue 1 2009Wei Yang Seow A new class of triblock oligopeptides, containing arginine for DNA binding, histidine for intracellular buffering, and hydrophobic residues for enhanced cellular uptake has been designed, with each block offering unique functionalities essential for efficient gene delivery. Together, these materials demonstrate strong DNA binding ability, low cytotoxicity, and significantly higher in vivo gene-transfection efficiency compared to the PEI standard. [source] Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activityJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 1 2005Jianya Y Huan Abstract Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the ,-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the ,empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure. Copyright © 2004 Society of Chemical Industry [source] Analysis and prediction of protein folding rates using quadratic response surface modelsJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 10 2008Liang-Tsung Huang Abstract Understanding the relationship between amino acid sequences and folding rates of proteins is an important task in computational and molecular biology. In this work, we have systematically analyzed the composition of amino acid residues for proteins with different ranges of folding rates. We observed that the polar residues, Asn, Gln, Ser, and Lys, are dominant in fast folding proteins whereas the hydrophobic residues, Ala, Cys, Gly, and Leu, prefer to be in slow folding proteins. Further, we have developed a method based on quadratic response surface models for predicting the folding rates of 77 two- and three-state proteins. Our method showed a correlation of 0.90 between experimental and predicted protein folding rates using leave-one-out cross-validation method. The classification of proteins based on structural class improved the correlation to 0.98 and it is 0.99, 0.98, and 0.96, respectively, for all-,, all-,, and mixed class proteins. In addition, we have utilized Baysean classification theory for discriminating two- and three-state proteins, which showed an accuracy of 90%. We have developed a web server for predicting protein folding rates and it is available at http://bioinformatics.myweb.hinet.net/foldrate.htm. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2008 [source] Computational alanine scanning of the 1:1 human growth hormone,receptor complexJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 1 2002Shuanghong Huo Abstract The MM-PBSA (Molecular Mechanics,Poisson,Boltzmann surface area) method was applied to the human Growth Hormone (hGH) complexed with its receptor to assess both the validity and the limitations of the computational alanine scanning approach. A 400-ps dynamical trajectory of the fully solvated complex was simulated at 300 K in a 101 Å×81 Å×107 Å water box using periodic boundary conditions. Long-range electrostatic interactions were treated with the particle mesh Ewald (PME) summation method. Equally spaced snapshots along the trajectory were chosen to compute the binding free energy using a continuum solvation model to calculate the electrostatic desolvation free energy and a solvent-accessible surface area approach to treat the nonpolar solvation free energy. Computational alanine scanning was performed on the same set of snapshots by mutating the residues in the structural epitope of the hormone and the receptor to alanine and recomputing the ,Gbinding. To further investigate a particular structure, a 200-ps dynamical trajectory of an R43A hormone,receptor complex was simulated. By postprocessing a single trajectory of the wild-type complex, the average unsigned error of our calculated ,,Gbinding is ,1 kcal/mol for the alanine mutations of hydrophobic residues and polar/charged residues without buried salt bridges. When residues involved in buried salt bridges are mutated to alanine, it is demonstrated that a separate trajectory of the alanine mutant complex can lead to reasonable agreement with experimental results. Our approach can be extended to rapid screening of a variety of possible modifications to binding sites. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 15,27, 2002 [source] Modular, self-assembling peptide linkers for stable and regenerable carbon nanotube biosensor interfaces,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006Mark R. Contarino Abstract As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1,mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15,nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain. Copyright © 2006 John Wiley & Sons, Ltd. [source] Characterization of prostate-specific antigen binding peptides selected by phage display technologyJOURNAL OF MOLECULAR RECOGNITION, Issue 1 2006Catherine Ferrieu-Weisbuch Abstract Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82,87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer. Copyright © 2005 John Wiley & Sons, Ltd. [source] Simulation of the N -terminus of HIV-1 glycoprotein 41000 fusion peptide in micellesJOURNAL OF PEPTIDE SCIENCE, Issue 4 2005Allison Langham Abstract In this paper, the N -terminus of glycoprotein-41, the HIV-1 fusion peptide, was studied by molecular dynamics simulations in an explicit sodium dodecyl sulfate micelle. The simulation provides a detailed picture of the equilibrium structure and peptide stability as it interacts with the micelle. The equilibrium location of the peptide shows the peptide at the surface of the micelle with hydrophobic residues interacting with the micelle's core. At equilibrium, the peptide adopts an ,-helical structure from residues 5,16 and a type-1 ,-turn from 17,20 with the other residues exhibiting more flexible conformations. The primary hydrophobic interactions with the micelle are from the leucine and phenylalanine residues (Leu-7, Phe-8, Leu-9, Phe-11, Leu-12) while the alanine and glycine residues (Ala-1, Gly-3, Gly-5, Ala-6, Gly-10, Gly-13, Ala-14, Ala-15, Gly-16, Gly-10, Ala-21) interact favorably with water molecules. The results suggest that Phe-8, part of the highly conserved FLG motif of the fusion peptide, plays a key role in the interaction of the peptide with membranes. Our simulations corroborate experimental investigations of the fusion peptide in SDS micelles, providing a high-resolution picture that explains the experimental findings. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd. [source] Sustained and Extended Release with Structural and Activity Recovery of Lysozyme from Complexes with Sodium (Sulfamate Carboxylate) Isoprene/Ethylene Oxide Block CopolymerMACROMOLECULAR BIOSCIENCE, Issue 2 2010Gao Gao Abstract The complexation of lysozyme and sodium (sulfamate carboxylate) isoprene/ethylene oxide (SCIEO) at pH,=,7.4 and the release of lysozyme from the complexes in the presence of NaCl were investigated. Through electrostatic and hydrophobic interactions, lysozyme and SCIEO form stable complex nanoparticles. The complexation partially disturbs the structure of lysozyme. Some of the hydrophobic residues of lysozyme are exposed to bind with SCIEO. The complexation leads to loss of most of the lysozyme activity. In the presence of NaCl, lysozyme can be released from the complexes. The released lysozyme molecules recover their native structure and activity completely. In the condition of physiological pH and ionic strength, a sustained and extended release of lysozyme was achieved. [source] Evidence for the adaptive evolution of the carbon fixation gene rbcL during diversification in temperature tolerance of a clade of hot spring cyanobacteriaMOLECULAR ECOLOGY, Issue 5 2003S. R. Miller Abstract Determining the molecular basis of enzyme adaptation is central to understanding the evolution of environmental tolerance but is complicated by the fact that not all amino acid differences between ecologically divergent taxa are adaptive. Analysing patterns of nucleotide sequence evolution can potentially guide the investigation of protein adaptation by identifying candidate codon sites on which diversifying selection has been operating. Here, I test whether there is evidence for molecular adaptation of the carbon fixation gene rbcL for a clade of hot spring cyanobacteria in the genus Synechococcus that has diverged in thermotolerance. Amino acid replacements during Synechococcus radiation have resulted in an increase in the number of hydrophobic residues in the RbcLs of more thermotolerant strains. A similar increase in hydrophobicity has been observed for many thermostable proteins. Maximum likelihood models which allow for heterogeneity among codon sites in the ratio of nonsynonymous to synonymous nucleotide substitutions estimated a class of amino acid sites as a target of positive selection. Depending on the model, a single amino acid site that interacts with a flexible element involved in the opening and closing of the active site was estimated with either low or moderate support to be a member of this class. Site-directed mutagenesis approaches are being explored in order to directly test its adaptive significance. [source] Interaction Between N-terminal Loop and , -Scaffold of Photoactive Yellow Protein,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Miki Harigai During the photoreaction cycle of photoactive yellow protein (PYP), a physiologically active intermediate (PYPM) is formed as a consequence of global protein conformational change. Previous studies have demonstrated that the photocycle of PYP is regulated by the N-terminal loop region, which is located across the central , -sheet from the p -coumaric acid chromophore. In this paper, the hydrophobic interaction between N-terminal loop and , -sheet was studied by characterizing PYP mutants of the hydrophobic residues. The rate constants and structural changes of the photocycle of L15A and L23A possibly participating in such an interaction were more similar to wild-type than F6A, showing that the CH/, interaction between Phe6 and Lys123 is the most essential as reported previously. To better understand the interactions between N-terminal tail and , -sheet of PYP, Phe6 and Phe121 were replaced by Cys and linked by a disulfide bond. Since the photocycle kinetics, structural change and thermal stability of F6C/F121C were similar to F6A, the CH/, interaction between Phe6 and Lys123 is not substitutable. It is likely that the detachment of position 6 from position 123 substantially alters the nature of PYP. [source] Active TEM-1 ,-lactamase mutants with random peptides inserted in three contiguous surface loopsPROTEIN SCIENCE, Issue 10 2006Pascale Mathonet Abstract Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 ,-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two ,-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first ,-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final ,-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium-sized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified. [source] The MRG domain of human MRG15 uses a shallow hydrophobic pocket to interact with the N-terminal region of PAM14PROTEIN SCIENCE, Issue 10 2006Peng Zhang Abstract MRG15 is a transcription factor expressed in a variety of human tissues, and its orthologs have been found in many other eukaryotes which constitute the MRG protein family. It plays a vital role in embryonic development and cell proliferation, and is involved in cellular senescence. The C-terminal part of MRG15 forms a conserved MRG domain which is involved in interactions with the tumor suppressor protein retinoblastoma and a nucleoprotein PAM14 during transcriptional regulation. We report here the characterization of the interaction between the MRG domain of human MRG15 and PAM14 using both yeast two-hybrid and in vitro binding assays based on the crystal structure of the MRG domain. The MRG domain is predominantly hydrophobic, and consists of mainly ,-helices that are arranged in a three-layer sandwich topology. The hydrophobic core is stabilized by interactions among a number of conserved hydrophobic residues. The molecular surface is largely hydrophobic, but contains a few hydrophilic patches. Structure-based site-directed mutagenesis studies identified key residues involved in the binding of PAM14. Structural and biochemical data together demonstrate that the PAM14 binding site is consisted of residues Ile160, Leu168, Val169, Trp172, Tyr235, Val268, and Arg269 of MRG15, which form a shallow hydrophobic pocket to interact with the N-terminal 50 residues of PAM14 through primarily hydrophobic interactions. These results provide the molecular basis for the interaction between the MRG domain and PAM14, and reveal insights into the potential biological function of MRG15 in transcription regulation and chromatin remodeling. [source] Frequencies of hydrophobic and hydrophilic runs and alternations in proteins of known structurePROTEIN SCIENCE, Issue 1 2006Russell Schwartz Abstract Patterns of alternation of hydrophobic and polar residues are a profound aspect of amino acid sequences, but a feature not easily interpreted for soluble proteins. Here we report statistics of hydrophobicity patterns in proteins of known structure in a current protein database as compared with results from earlier, more limited structure sets. Previous studies indicated that long hydrophobic runs, common in membrane proteins, are underrepresented in soluble proteins. Long runs of hydrophobic residues remain significantly underrepresented in soluble proteins, with none longer than 16 residues observed. These long runs most commonly occur as buried , helices, with extended hydrophobic strands less common. Avoiding aggregation of partially folded intermediates during intracellular folding remains a viable explanation for the rarity of long hydrophobic runs in soluble proteins. Comparison between database editions reveals robustness of statistics on aqueous proteins despite an approximately twofold increase in nonredundant sequences. The expanded database does now allow us to explain several deviations of hydrophobicity statistics from models of random sequence in terms of requirements of specific secondary structure elements. Comparison to prior membrane-bound protein sequences, however, shows significant qualitative changes, with the average hydrophobicity and frequency of long runs of hydrophobic residues noticeably increasing between the database editions. These results suggest that the aqueous proteins of solved structure may represent an essentially complete sample of the universe of aqueous sequences, while the membrane proteins of known structure are not yet representative of the universe of membrane-associated proteins, even by relatively simple measures of hydrophobic patterns. [source] Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor HPROTEIN SCIENCE, Issue 5 2004Lena Kask C4BP, C4b-binding protein; FH, factor H; CCP, complement control protein; CD, circular dichroism; FTIR, Fourier transform-infrared spectroscopy; PT, prothrombin; VCP, vaccinia virus complement control protein Abstract The complement inhibitors C4b-binding protein (C4BP) and factor H (FH) both consist of complement control protein (CCP) domains. Here we examined the secondary structure of both proteins by circular dichroism and Fourier-transform infrared technique at temperatures ranging from 30°C,90°C. We found that predominantly ,-sheet structure of both proteins was stable up to 70°C, and that a reversible conformational change toward ,-helix was apparent at temperatures ranging from 70°C to 90°C. The ability of both proteins to inhibit complement was not impaired after incubation at 95°C, exposure to extreme pH conditions, and storage at room temperature for several months. Similar remarkable stability was previously observed for vaccinia virus control protein (VCP), which is also composed of CCP domains; it therefore seems to be a general property of CCP-containing proteins. A typical CCP domain has a hydrophobic core, which is wrapped in ,-sheets and stabilized by two disulphide bridges. How the CCP domains tolerate harsh conditions is unclear, but it could be due to a combination of high content of prolines, hydrophobic residues, and the presence of two disulphide bridges within each domain. These findings are of interest because CCP-containing complement inhibitors have been proposed as clinical agents to be used to control unwanted complement activation that contributes to many diseases. [source] Effects of denaturants and substitutions of hydrophobic residues on backbone dynamics of denatured staphylococcal nucleasePROTEIN SCIENCE, Issue 7 2003Satoshi Ohnishi NOE, nuclear Overhauser effect; RDC, residual dipolar coupling Abstract Analysis of residual dipolar couplings (RDCs) in the ,131, fragment of staphylococcal nuclease has demonstrated that its ensemble-averaged structure is resistant to perturbations such as high concentrations of urea, low pH, and substitution of hydrophobic residues, suggesting that its residual structure is encoded by local side-chain/backbone interactions. In the present study, the effects of these same perturbations on the backbone dynamics of ,131, were examined through 1H- 15N relaxation methods. Unlike the global structure reported by RDCs, the transverse relaxation rates R2 were quite sensitive to denaturing conditions. At pH 5.2, ,131, exhibits an uneven R2 profile with several characteristic peaks involving hydrophobic chain segments. Protonation of carboxyl side chains by lowering the pH reduces the values of R2 along the entire chain, yet these characteristic peaks remain. In contrast, high concentrations of urea or the substitution of 10 hydrophobic residues eliminates these peaks and reduces the R2 values by a greater amount. The combination of low pH and high urea leads to further decreases in R2. These denaturant-induced increases in backbone mobility are also reflected in decreases in 15N NOEs and in relaxation interference parameters, with the former reporting an increase in fast motions and the latter a decrease in slow motions. Comparison between the changes in chain dynamics and the corresponding changes in Stokes radius and the patterns of RDCs suggests that regional variations in backbone dynamics in denatured nuclease arise primarily from local contacts between hydrophobic side chains and local interactions involving charged carboxyl groups. [source] A helix-turn motif in the C-terminal domain of histone H1PROTEIN SCIENCE, Issue 4 2000Roger Vila Abstract The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKT KKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone Hl° (residues 99,121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i, i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C, protons, span from Pro100 to Val 116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the ,-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) ,-turn conformation, a ,-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs. [source] Aromatic residues at position 55 of rat ,7 nicotinic acetylcholine receptors are critical for maintaining rapid desensitizationTHE JOURNAL OF PHYSIOLOGY, Issue 4 2008Elaine A. Gay The rat ,7 nicotinic acetylcholine receptor (nAChR) can undergo rapid onset of desensitization; however, the mechanisms of desensitization are largely unknown. The contribution of a tryptophan (W) residue at position 55 of the rat ,7 nAChR subunit, which lies within the ,2 strand, was studied by mutating it to other hydrophobic and/or aromatic amino acids, followed by voltage-clamp experiments in Xenopus oocytes. When mutated to alanine, the ,7-W55A nAChR desensitized more slowly, and recovered from desensitization more rapidly, than wildtype ,7 nAChRs. The contribution of desensitization was validated by kinetic modelling. Mutating W55 to other aromatic residues (phenylalanine or tyrosine) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (alanine, cysteine or valine) significantly decreased the rate of onset and increased the rate of recovery from desensitization. To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of W55 by mutating W55 to cysteine (,7-W55C) and testing the ability of various sulfhydryl reagents to react with this cysteine. Several positively charged sulfhydryl reagents blocked ACh-induced responses for ,7-W55C nAChRs, whereas a neutral sulfhydryl reagent potentiated responses; residue C55 was not accessible for modification in the desensitized state. These data suggest that W55 plays an important role in both the onset and recovery from desensitization in the rat ,7 nAChR, and that aromatic residues at position 55 are critical for maintaining rapid desensitization. Furthermore, these data suggest that W55 may be a potential target for modulatory agents operating via hydrophobic interactions. [source] ATP sensitivity of ATP-sensitive K+ channels: role of the , phosphate group of ATP and the R50 residue of mouse Kir6.2THE JOURNAL OF PHYSIOLOGY, Issue 3 2005Scott A. John ATP-sensitive K (KATP) channels are composed of Kir6, the pore-forming protein, and the sulphonylurea receptor SUR, a regulatory protein. We and others have previously shown that positively charged residues in the C terminus of Kir6.2, including R201 and K185, interact with the , and , phosphate groups of ATP, respectively, to induce channel closure. A positively charged residue in the N terminus, R50, is also important, and has been proposed to interact with either the , or , phosphate group of ATP. To examine this issue, we systematically mutated R50 to residues of different size, charge and hydropathy, and examined the effects on adenine nucleotide sensitivity in the absence and presence of SUR1. In the absence of SUR1, only the size of residue 50 significantly altered ATP sensitivity, with smaller side chains decreasing ATP sensitivity. In the presence of SUR1, however, hydrophathy and charge also played a role. Hydrophilic residues decreased ATP sensitivity more than hydrophobic residues for small size residues, and, surprisingly, negatively charged residues E and D preserved ATP sensitivity and increased ADP sensitivity relative to the wild-type residue R. These observations suggest that a negative charge near position 50, due to either mutation of R50 or the interaction of the , phosphate group of ATP with R50, facilitates closure of the ATP-dependent gate. Mutation of the nearby positively charged residue R54, known to be involved in stabilizing channel opening via electrostatic interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), also caused increased ADP sensitivity as compared with ATP, suggesting a loss of function of ATP's , phosphate. Based on these results, we propose that a phosphate group or a negative charge at position 50 initiates channel closure by destabilizing the electrostatic interactions between negative phosphate groups of PIP2 and residues such as R54. [source] The role of entropy and polarity in intermolecular contacts in protein crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Marcin Cie The integrity and X-ray diffraction quality of protein crystals depend on the three-dimensional order of relatively weak but reproducible intermolecular contacts. Despite their importance, relatively little attention has been paid to the chemical and physical nature of these contacts, which are often regarded as stochastic and thus not different from randomly selected protein surface patches. Here, logistic regression was used to analyze crystal contacts in a database of 821 unambiguously monomeric proteins with structures determined to 2.5,Å resolution or better. It is shown that the propensity of a surface residue for incorporation into a crystal contact is not a linear function of its solvent-accessible surface area and that amino acids with low exposed surfaces, which are typically small and hydrophobic, have been underestimated with respect to their contact-forming potential by earlier area-based calculations. For any given solvent-exposed surface, small and hydrophobic residues are more likely to be involved in crystal contacts than large and charged amino acids. Side-chain entropy is the single physicochemical property that is most negatively correlated with the involvement of amino acids in crystal contacts. It is also shown that crystal contacts with larger buried surfaces containing eight or more amino acids have cores that are depleted of polar amino acids. [source] Structure-assisted discovery of an aminothiazole derivative as a lead molecule for inhibition of bacterial fatty-acid synthesisACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2007Günter Pappenberger Fatty-acid synthesis in bacteria is of great interest as a target for the discovery of antibacterial compounds. The addition of a new acetyl moiety to the growing fatty-acid chain, an essential step in this process, is catalyzed by ,-ketoacyl-ACP synthase (KAS). It is inhibited by natural antibiotics such as cerulenin and thiolactomycin; however, these lack the requirements for optimal drug development. Structure-based biophysical screening revealed a novel synthetic small molecule, 2-phenylamino-4-methyl-5-acetylthiazole, that binds to Escherichia coli KAS I with a binding constant of 25,µM as determined by fluorescence titration. A 1.35,Å crystal structure of its complex with its target reveals noncovalent interactions with the active-site Cys163 and hydrophobic residues of the fatty-acid binding pocket. The active site is accessible through an open conformation of the Phe392 side chain and no conformational changes are induced at the active site upon ligand binding. This represents a novel binding mode that differs from thiolactomycin or cerulenin interaction. The structural information on the protein,ligand interaction offers strategies for further optimization of this low-molecular-weight compound. [source] The structure of human deoxycytidine kinase in complex with clofarabine reveals key interactions for prodrug activationACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2006Yan Zhang Clofarabine [2-chloro-9-(2-deoxy-2-fluoro-,- d -arabinofuranosyl)-9H -purin-6-amine] is a hybrid of the widely used anticancer drugs cladribine and fludarabine. It is the precursor of an effective chemotherapeutic agent for leukemias and other hematological malignancies and received accelerated approval by the FDA for the treatment of pediatric patients with relapsed or refractory acute lymphoblastic leukemia. Clofarabine is phosphorylated intracellularly by human deoxycytidine kinase (dCK) to the 5,-monophosphate, which is the rate-limiting step in activation of the prodrug. dCK has a broad substrate specificity, with a much higher activity to deoxycytidine than to deoxyadenosine and deoxyguanosine. As a purine-nucleoside analog, clofarabine is a better substrate of dCK than deoxycytidine. The crystal structure of dCK has been solved previously in complex with pyrimidine nucleosides and ADP [Sabini et al. (2003), Nature Struct. Biol.10, 513,519]. In the current study, the crystal structure of clofarabine- and ADP-bound dCK was solved to 2.55,Å by molecular replacement. It appears that the enzyme takes the same conformation as in the structures of the pyrimidine nucleoside-bound complexes. The interactions between 2-Cl and its surrounding hydrophobic residues contribute to the high catalytic efficiency of dCK for clofarabine. [source] Structure of human glycolate oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazoleACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Jean-Marie Bourhis Glycolate oxidase, a peroxisomal flavoenzyme, generates glyoxylate at the expense of oxygen. When the normal metabolism of glyoxylate is impaired by the mutations that are responsible for the genetic diseases hyperoxaluria types 1 and 2, glyoxylate yields oxalate, which forms insoluble calcium deposits, particularly in the kidneys. Glycolate oxidase could thus be an interesting therapeutic target. The crystal structure of human glycolate oxidase (hGOX) in complex with 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST) has been determined at 2.8,Å resolution. The inhibitor heteroatoms interact with five active-site residues that have been implicated in catalysis in homologous flavodehydrogenases of l -2-hydroxy acids. In addition, the chlorophenyl substituent is surrounded by nonconserved hydrophobic residues. The present study highlights the role of mobility in ligand binding by glycolate oxidase. In addition, it pinpoints several structural differences between members of the highly conserved family of flavodehydrogenases of l -2-hydroxy acids. [source] Structure of the adenylation domain of NAD+ -dependent DNA ligase from Staphylococcus aureusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Seungil Han DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5,-phosphate and 3,-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD+ -dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD+ -dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD+ -binding pocket and the `C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors. [source] Structures of BIR domains from human NAIP and cIAP2ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Maria Dolores Herman The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1,,P4, side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3, position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2, and P4, pockets make similar interactions to those seen in other BIR domain,peptide complexes. The structures also reveal how a serine in the P1, position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins. [source] Disperse distribution of cationic amino acids on hydrophilic surface of helical wheel enhances antimicrobial peptide activityBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Young Soo Kim Abstract The antimicrobial action of amphipathic antimicrobial peptides (AMPs) generally depends on perturbation of the bacterial membrane via electrostatic interactions promoting initial binding to the surface and hydrophobic interactions for pore formation into the membrane. Several studies have focused on the structure,activity relationship (SAR) of AMPs by modulation of structural parameters. However, modulation of one parameter commonly induces simultaneous changes in other parameters, making it difficult to investigate the specific influence of a single variable. In the present work, we investigated the distribution effect of cationic amino acids on the hydrophilic surface of the helical wheel using model AMPs composed of only lysine (K) and leucine (L) as representative cationic and hydrophobic residues, respectively, under conditions in which other parameters are fixed. Based on SAR analyses of ,-helical KL model AMPs displaying different cationic distributions, we propose that the dispersity of cationic amino acids on the hydrophilic surface is a factor that contributes to the antimicrobial activity of AMP. Moreover, antimicrobial activity is enhanced by rearrangement of cationic amino acids to promote dispersed distribution. We confirmed the cationic distribution effect using natural AMP-derived ,-helical CRAMP18 and its analogs. Our data show that accumulation of lysine shifts in the CRAMP18 analog leads to higher dispersion, and subsequently to improved antimicrobial activity. Therefore, we propose that the cationic distribution effect can be applied for the rational redesign of amino acid sequences to improve the antimicrobial activities of natural ,-helical AMPs, in combination with regulation of other known structural parameters. Biotechnol. Bioeng. 2010;107: 216,223. © 2010 Wiley Periodicals, Inc. [source] | ||||||||||||||||