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Hydrophobic Core (hydrophobic + core)
Selected AbstractsHydrophobically Directed Aldol Reactions: Polystyrene-Supported L -Proline as a Recyclable Catalyst for Direct Asymmetric Aldol Reactions in the Presence of Water,EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 28 2007Michelangelo Gruttadauria Abstract A simple synthetic methodology for the preparation of a polystyrene-supported L -proline material is reported, and this material has been used as catalyst in direct asymmetric aldol reactions between several ketones and arylaldehydes to furnish aldol products in high yields and stereoselectivities. Screening of solvents showed that these reactions take place only in the presence of water or methanol, at lower levels of conversion in the latter case. This solvent effect, coupled with the observed high stereoselectivities, has been explained in terms of the formation of a hydrophobic core in the inner surface of the resin, whereas the hydrophilic proline moiety lies at the resin/water interface. Such a microenvironment both promotes the aldol reaction and increases the stereoselectivity. Recycling investigations have shown that this material can be reused, without loss in levels of conversion and stereoselectivity, for at least five cycles. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] The stop transfer sequence of the human UDP-glucuronosyltransferase 1A determines localization to the endoplasmic reticulum by both static retention and retrieval mechanismsFEBS JOURNAL, Issue 4 2005Lydia Barré Human UDP-glucuronosyltransferase 1A (UGT1A) isoforms are endoplasmic reticulum (ER)-resident type I membrane proteins responsible for the detoxification of a broad range of toxic phenolic compounds. These proteins contain a C-terminal stop transfer sequence with a transmembrane domain (TMD), which anchors the protein into the membrane, followed by a short cytosolic tail (CT). Here, we investigated the mechanism of ER residency of UGT1A mediated by the stop transfer sequence by analysing the subcellular localization and sensitivity to endoglycosidases of chimeric proteins formed by fusion of UGT1A stop transfer sequence (TMD/CT) with the ectodomain of the plasma membrane CD4 reporter protein. We showed that the stop transfer sequence, when attached to C-terminus of the CD4 ectodomain was able to prevent it from being transported to the cell surface. The protein was retained in the ER indicating that this sequence functions as an ER localization signal. Furthermore, we demonstrated that ER localization conferred by the stop transfer sequence was mediated in part by the KSKTH retrieval signal located on the CT. Interestingly, our data indicated that UGT1A TMD alone was sufficient to retain the protein in ER without recycling from Golgi compartment, and brought evidence that organelle localization conferred by UGT1A TMD was determined by the length of its hydrophobic core. We conclude that both retrieval mechanism and static retention mediated by the stop transfer sequence contribute to ER residency of UGT1A proteins. [source] Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopiesFEBS JOURNAL, Issue 22 2003Tanya Sheynis Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid,water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides. [source] Mapping the functional domain of the prion proteinFEBS JOURNAL, Issue 16 2003Taian Cui Prion diseases such as Creutzfeldt,Jakob disease are possibly caused by the conversion of a normal cellular glycoprotein, the prion protein (PrPc) into an abnormal isoform (PrPSc). The process that causes this conversion is unknown, but to understand it requires a detailed insight into the normal activity of PrPc. It has become accepted from results of numerous studies that PrPc is a Cu-binding protein and that its normal function requires Cu. Further work has suggested that PrPc is an antioxidant with an activity like that of a superoxide dismutase. We have shown in this investigation that this activity is optimal for the whole protein and that deletion of parts of the protein reduce or abolish this activity. The protein therefore contains an active domain requiring certain regions such as the Cu-binding octameric repeat region and the hydrophobic core. These regions show high evolutionary conservation fitting with the idea that they are important to the active domain of the protein. [source] Bionanotechnology: Enhancement of Aggregation-Induced Emission in Dye-Encapsulating Polymeric Micelles for Bioimaging (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 9 2010Mater. Amphiphilic block copolymers can form polymer micelles for delivering hydrophobic fluorescent probes with aggregation-induced emission properties, as presented by A. K.-Y. Jen et al. on page 1413. By itself, 1,1,2,3,4,5-hexaphenylsilole (HPS) exhibits dramatically enhanced blue-green fluorescent emission efficiencies when encapsulated within the hydrophobic core of a polymeric micelle. When HPS is co-encapsulated with bis(4-(N -(1-naphthyl) phenylamino)-phenyl)fumaronitrile, effective orange-red fluorescence resonance energy transfer can be demonstrated within live RAW 264.7 cells. Illustration provided by Brent Polishak. [source] Enhancement of Aggregation-Induced Emission in Dye-Encapsulating Polymeric Micelles for BioimagingADVANCED FUNCTIONAL MATERIALS, Issue 9 2010Wen-Chung Wu Abstract Three amphiphilic block copolymers are employed to form polymeric micelles and function as nanocarriers to disperse hydrophobic aggregation-induced emission (AIE) dyes, 1,1,2,3,4,5-hexaphenylsilole (HPS) and/or bis(4-(N -(1-naphthyl) phenylamino)-phenyl)fumaronitrile (NPAFN), into aqueous solution for biological studies. Compared to their virtually non-emissive properties in organic solutions, the fluorescence intensity of these AIE dyes has increased significantly due to the spatial confinement that restricts intramolecular rotation of these dyes and their better compatibility in the hydrophobic core of polymeric micelles. The effect of the chemical structure of micelle cores on the photophysical properties of AIE dyes are investigated, and the fluorescence resonance energy transfer (FRET) from the green-emitting donor (HPS) to the red-emitting acceptor (NPAFN) is explored by co-encapsulating this FRET pair in the same micelle core. The highest fluorescence quantum yield (,62%) could be achieved by encapsulating HPS aggregates in the micelles. Efficient energy transfer (>99%) and high amplification of emission (as high as 8 times) from the NPAFN acceptor could also be achieved by spatially confining the HPS/NPAFN FRET pair in the hydrophobic core of polymeric micelles. These micelles could be successfully internalized into the RAW 264.7 cells to demonstrate high-quality fluorescent images and cell viability due to improved quantum yield and reduced cytotoxicity. [source] Molecular characterization of novel progranulin (GRN) mutations in frontotemporal dementia,HUMAN MUTATION, Issue 4 2008Odity Mukherjee Abstract Frontotemporal dementia (FTD) is a clinical term encompassing dementia characterized by the presence of two major phenotypes: 1) behavioral and personality disorder, and 2) language disorder, which includes primary progressive aphasia and semantic dementia. Recently, the gene for familial frontotemporal lobar degeneration (FTLD) with ubiquitin-positive, tau-negative inclusions (FTLD-U) linked to chromosome 17 was cloned. In the present study, 62 unrelated patients from the Washington University Alzheimer's Disease Research Center and the Midwest Consortium for FTD with clinically diagnosed FTD and/or neuropathologically characterized cases of FTLD-U with or without motor neuron disease (MND) were screened for mutations in the progranulin gene (GRN; also PGRN). We discovered two pathogenic mutations in four families: 1) a single-base substitution within the 3, splice acceptor site of intron 6/exon 7 (g.5913A>G [IVS6,2A>G]) causing skipping of exon 7 and premature termination of the coding sequence (PTC); and 2) a missense mutation in exon 1 (g.4068C>A) introducing a charged amino acid in the hydrophobic core of the signal peptide at residue 9 (p.A9D). Functional analysis in mutation carriers for the splice acceptor site mutation revealed a 50% decrease in GRN mRNA and protein levels, supporting haploinsufficiency. In contrast, there was no significant difference in the total GRN mRNA between cases and controls carrying the p.A9D mutation. Further, subcellular fractionation and confocal microscopy indicate that although the mutant protein is expressed, it is not secreted, and appears to be trapped within an intracellular compartment, possibly resulting in a functional haploinsufficiency. Hum Mutat 29(4), 512,521, 2008. © 2008 Wiley-Liss, Inc. [source] Preparation and in vitro release of D,L -tetrahydropalmatine-loaded graft copolymer nanoparticlesJOURNAL OF APPLIED POLYMER SCIENCE, Issue 6 2008Yinglei Zhai Abstract D,L -tetrahydropalmatine (THP)-loaded poly{[,-maleic anhydride-,-methoxy-poly(ethylene glycol)]- co -(ethy cyanoacrylate)} (PEGECA) amphiphilic graft copolymer nanoparticles (PEGECAT NPs) were prepared by the nanoprecipitation technique. The effects of solvent property, temperature, copolymer composition, and drug feeding on the drug-loaded amount and size of PEGECAT NPs were investigated. The morphological structure of PEGECAT NPs was characterized by transmission electron microscopy (TEM), proton nuclear magnetic resonance (1H NMR), and the size was measured by laser particle size analyzer (LPSA). In vitro release behaviors of drug from PEGECAT NPs were examined by high-pressure liquid chromatography (HPLC). The results demonstrate that PEGECAT NPs take on a spherical morphology with an inner core and outer shell before and after in vitro release. THP can be incorporated into the hydrophobic core of PEGECAT NPs and the drug-loaded amount is higher than 5%. The release of THP from PEGECAT NPs is initially fast and then slows down. The accumulated release is lower than 40% after 48 h. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] Interaction of a ,-sheet breaker peptide with lipid membranesJOURNAL OF PEPTIDE SCIENCE, Issue 2 2010Giuseppe Vitiello Abstract Aggregation of ,-amyloid peptides into senile plaques has been identified as one of the hallmarks of Alzheimer's disease. An attractive therapeutic strategy for Alzheimer's disease is the inhibition of the soluble ,-amyloid aggregation using synthetic ,-sheet breaker peptides that are capable of binding A, but are unable to become part of a ,-sheet structure. As the early stages of the A, aggregation process are supposed to occur close to the neuronal membrane, it is strategic to define the ,-sheet breaker peptide positioning with respect to lipid bilayers. In this work, we have focused on the interaction between the ,-sheet breaker peptide acetyl-LPFFD-amide, iA,5p, and lipid membranes, studied by ESR spectroscopy, using either peptides alternatively labeled at the C- and at the N-terminus or phospholipids spin-labeled in different positions of the acyl chain. Our results show that iA,5p interacts directly with membranes formed by the zwitterionic phospholipid dioleoyl phosphatidylcholine and this interaction is modulated by inclusion of cholesterol in the lipid bilayer formulation, in terms of both peptide partition coefficient and the solubilization site. In particular, cholesterol decreases the peptide partition coefficient between the membrane and the aqueous medium. Moreover, in the absence of cholesterol, iA,5p is located between the outer part of the hydrophobic core and the external hydrophilic layer of the membrane, while in the presence of cholesterol it penetrates more deeply into the lipid bilayer. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. [source] The Use of the RAFT-Technique for the Preparation of Temperature/pH Sensitive Polymers in Different ArchitecturesMACROMOLECULAR SYMPOSIA, Issue 1 2009Angel Licea-Claveríe Abstract In this contribution we report the use of the RAFT-technique for the preparation of three types of responsive polymeric materials with a high potential of application in the biomedical field: 1.-Diblock copolymers with reversible self-assembly capacity as a function of pH based on N,N, -diethylaminoethyl methacrylate (DEAEM) and 2-methacryloyloxy benzoic acid (MAOB); 2.-Diblock copolymers with reversible self-assembly capacity as a function of temperature, based N -isopropylacrylamide (NIPAAm) and n-hexyl acrylate (HA); and 3.-Polymeric stars with random number of arms consisting either in NIPAAm-arms or copolymeric NIPAAm-arms and hydrophobic core. [source] Structure elucidation and 3D solution conformation of the antibiotic enduracidin determined by NMR spectroscopy and molecular dynamicsMAGNETIC RESONANCE IN CHEMISTRY, Issue 8 2005F. Castiglione Abstract Enduracidin and ramoplanin belong to the large family of cyclodepsipeptide antibiotics, highly effective against Gram-positive bacteria. The primary and 3D solution structure of ramoplanin is already well known, and the primary structure of enduracidin has been determined by a combination of chemical and NMR spectroscopic methods. Both antibiotics share a similar peptide core of 17 amino acids and differ mainly in the length of the acyl chain and the presence of two D -mannose moieties in ramoplanin. Based on the high sequence homology with ramoplanin, the structure in solution of enduracidin is modeled as a cyclic peptide. The tertiary structure thus obtained was refined through molecular dynamics (MD) simulation, in which the interatomic NOE-derived distance restraints were imposed. MD simulations yielded a family of representative 3D structures (RMSD = 0.89), which highlighted a backbone geometry similar to that of ramoplanin in its ,-hairpin arrangement. In contrast, enduracidin displays a different arrangement of the side-chain and of the residues forming the hydrophobic core. Copyright © 2005 John Wiley & Sons, Ltd. [source] Liposome/water lipophilicity: Methods, information content, and pharmaceutical applicationsMEDICINAL RESEARCH REVIEWS, Issue 3 2004Georgette Plemper van Balen Abstract This review discusses liposome/water lipophilicity in terms of the structure of liposomes, experimental methods, and information content. In a first part, the structural properties of the hydrophobic core and polar surface of liposomes are examined in the light of potential interactions with solute molecules. Particular emphasis is placed on the physicochemical properties of polar headgroups of lipids in liposomes. A second part is dedicated to three useful methods to study liposome/water partitioning, namely potentiometry, equilibrium dialysis, and 1H-NMR relaxation rates. In each case, the principle and limitations of the method are discussed. The next part presents the structural information encoded in liposome/water lipophilicity, in other words the solutes' structural and physicochemical properties that determine their behavior and hence their partitioning in such systems. This presentation is based on a comparison between isotropic (i.e., solvent/water) and anisotropic (e.g., liposome/water) systems. An important factor to be considered is whether the anisotropic lipid phase is ionized or not. Three examples taken from the authors' laboratories are discussed to illustrate the factors or combinations thereof that govern liposome/water lipophilicity, namely (a) hydrophobic interactions alone, (b) hydrophobic and polar interactions, and (c) conformational effects plus hydrophobic and ionic interactions. The next part presents two studies taken from the field of QSAR to exemplify the use of liposome/water lipophilicity in structure,disposition and structure,activity relationships. In the conclusion, we summarize the interests and limitations of this technology and point to promising developments. © 2004 Wiley Periodicals, Inc. Med Res Rev, 24, No. 3, 299,324, 2004 [source] Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineeringPROTEIN SCIENCE, Issue 8 2006Koki Makabe Abstract Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" ,-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Å resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel ,-sheet. These well-defined water molecules provide additional structural links between adjacent ,-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA. [source] Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDSPROTEIN SCIENCE, Issue 7 2006Junghwa Kim Abstract Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS,gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers. [source] In silico protein design by combinatorial assembly of protein building blocksPROTEIN SCIENCE, Issue 10 2004Hui-Hsu (Gavin) Tsai Abstract Utilizing concepts of protein building blocks, we propose a de novo computational algorithm that is similar to combinatorial shuffling experiments. Our goal is to engineer new naturally occurring folds with low homology to existing proteins. A selected protein is first partitioned into its building blocks based on their compactness, degree of isolation from the rest of the structure, and hydrophobicity. Next, the protein building blocks are substituted by fragments taken from other proteins with overall low sequence identity, but with a similar hydrophobic/hydrophilic pattern and a high structural similarity. These criteria ensure that the designed protein has a similar fold, low sequence identity, and a good hydrophobic core compared with its native counterpart. Here, we have selected two proteins for engineering, protein G B1 domain and ubiquitin. The two engineered proteins share ,20% and ,25% amino acid sequence identities with their native counterparts, respectively. The stabilities of the engineered proteins are tested by explicit water molecular dynamics simulations. The algorithm implements a strategy of designing a protein using relatively stable fragments, with a high population time. Here, we have selected the fragments by searching for local minima along the polypeptide chain using the protein building block model. Such an approach provides a new method for engineering new proteins with similar folds and low homology. [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] The role of strand 1 of the C ,-sheet in the structure and function of ,1 -antitrypsinPROTEIN SCIENCE, Issue 12 2001Stephen P. Bottomley Abstract Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C ,-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant ,1 -antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10, position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7, and P9, caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6, and P8, positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6, and P8, residues of the C ,-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6, and P8, mutants of ,1 -antitrypsin had melting temperatures 14 degrees lower than wild-type ,1 -antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants. [source] The identification of conserved interactions within the SH3 domain by alignment of sequences and structuresPROTEIN SCIENCE, Issue 11 2000Stefan M. Larson Abstract The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein,protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide-binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side-chain to main-chain hydrogen bonds were observed in the functionally crucial RT-Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved ,-bulge was identified in the second ,-strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold. [source] The acid-induced folded state of Sac7d is the native statePROTEIN SCIENCE, Issue 10 2000Jennifer L. Bedell Abstract Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J, Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203,224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H, 15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid. [source] Structural plasticity of peanut lectin: an X-ray analysis involving variation in pH, ligand binding and crystal structureACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2004S. Kundhavai Natchiar Until recently, it has only been possible to grow crystals of peanut lectin when complexed with sugar ligands. It is now shown that it is possible to grow peanut lectin crystals at acidic pH in the presence of oligopeptides corresponding to a loop in the lectin molecule. Crystals have also been prepared in the presence of these peptides as well as lactose. Low-pH crystal forms of the lectin,lactose complex similar to those obtained at neutral pH have also been grown. Thus, crystals of peanut lectin grown under different environmental conditions, at two pH values with and without sugar bound to the lectin, are now available. They have been used to explore the plasticity and hydration of the molecule. A detailed comparison between different structures shows that the lectin molecule is sturdy and that the effect of changes in pH, ligand binding and environment on it is small. The region involving the curved front ,-sheet and the loops around the second hydrophobic core is comparatively rigid. The back ,-sheet involved in quaternary association, which exhibits considerable variability, is substantially flexible, as is the sugar-binding region. The numbers of invariant water molecules in the hydration shell are small and they are mainly involved in metal coordination or in stabilizing unusual structural features. Small consistent movements occur in the combining site upon sugar binding, although the site is essentially preformed. [source] High-resolution structure of an ,-spectrin SH3-domain mutant with a redesigned hydrophobic coreACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Ana Cámara-Artigas The ,-spectrin SH3 domain (Spc-SH3) is a small modular domain which has been broadly used as a model protein in folding studies and these studies have sometimes been supported by structural information obtained from the coordinates of Spc-SH3 mutants. The structure of B5/D48G, a multiple mutant designed to improve the hydrophobic core and as a consequence the protein stability, has been solved at 1,Å resolution. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 24.79, b = 37.23, c = 62.95,Å. This mutant also bears a D48G substitution in the distal loop and this mutation has also been reported to increase the stability of the protein by itself. The structure of the B5/D48G mutant shows a highly packed hydrophobic core and a more ordered distal loop compared with previous Spc-SH3 structures. [source] The structures of mutant forms of Hfq from Pseudomonas aeruginosa reveal the importance of the conserved His57 for the protein hexamer organizationACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Olga Moskaleva The bacterial Sm-like protein Hfq forms homohexamers both in solution and in crystals. The monomers are organized as a continuous ,-sheet passing through the whole hexamer ring with a common hydrophobic core. Analysis of the Pseudomonas aeruginosa Hfq (PaeHfq) hexamer structure suggested that solvent-inaccessible intermonomer hydrogen bonds created by conserved amino-acid residues should also stabilize the quaternary structure of the protein. In this work, one such conserved residue, His57, in PaeHfq was replaced by alanine, threonine or asparagine. The crystal structures of His57Thr and His57Ala Hfq were determined and the stabilities of all of the mutant forms and of the wild-type protein were measured. The results obtained demonstrate the great importance of solvent-inaccessible conserved hydrogen bonds between the Hfq monomers in stabilization of the hexamer structure. [source] A strained DNA binding helix is conserved for site recognition, folding nucleation, and conformational modulation,BIOPOLYMERS, Issue 6 2009Diana E. Wetzler Abstract Nucleic acid recognition is often mediated by ,-helices or disordered regions that fold into ,-helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical ,-helix is stabilized at the N-terminus, while the PII forms at the C-terminus half of the peptide. Re-examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N-terminus half is a canonical ,-helix, while the C-terminal half adopts a 310 helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 104 -fold lower affinity, and 500-fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained ,-helix conformation, "presented" by this unique architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 432,443, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Water Exclusion and Enantioselectivity in Catalysis,CHEMBIOCHEM, Issue 10 2006Ronald Breslow Prof. Dr. High and dry. Enzymes perform catalyses in the interior of the protein, which is not aqueous in character. Polyethylenimine enzyme mimics with a hydrophobic core also achieve high rates by excluding water. Two different approaches to the synthesis of such artificial enzymes with chirally attached side chains are described, as well as the use of these enzymes in performing biomimetic transaminations with chiral selectivity [source] Influence of the hydrophilic face on the folding ability and stability of ,-helix bundles: relevance to the peptide catalytic activityCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2000S.E. Blondelle Although not the sole feature responsible, the packing of amino acid side chains in the interior of proteins is known to contribute to protein conformational specificity. While a number of amphipathic peptide sequences with optimized hydrophobic domains has been designed to fold into a desired aggregation state, the contribution of the amino acids located on the hydrophilic side of such peptides to the final packing has not been investigated thoroughly. A set of self-aggregating 18-mer peptides designed previously to adopt a high level of ,-helical conformation in benign buffer is used here to evaluate the effect of the nature of the amino acids located on the hydrophilic face on the packing of a four ,-helical bundle. These peptides differ from one another by only one to four amino acid mutations on the hydrophilic face of the helix and share the same hydrophobic core. The secondary and tertiary structures in the presence or absence of denaturants were determined by circular dichroism in the far- and near-UV regions, fluorescence and nuclear magnetic resonance spectroscopy. Significant differences in folding ability, as well as chemical and thermal stabilities, were found between the peptides studied. In particular, surface salt bridges may form which would increase both the stability and extent of the tertiary structure of the peptides. The structural behavior of the peptides may be related to their ability to catalyze the decarboxylation of oxaloacetate, with peptides that have a well-defined tertiary structure acting as true catalysts. [source] A Designed Well-Folded Monomeric Four-Helix Bundle Protein Prepared by Fmoc Solid-Phase Peptide Synthesis and Native Chemical Ligation,CHEMISTRY - A EUROPEAN JOURNAL, Issue 5 2006Gunnar T. Dolphin Dr. Abstract The design and total chemical synthesis of a monomeric native-like four-helix bundle protein is presented. The designed protein, GTD-Lig, consists of 90 amino acids and is based on the dimeric structure of the de novo designed helix-loop-helix GTD-43. GTD-Lig was prepared by the native chemical ligation strategy and the fragments (45 residues long) were synthesized by applying standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The required peptide,thioester fragment was prepared by anchoring the free ,-carboxy group of Fmoc-Glu-allyl to the solid phase. After chain elongation the allyl moiety was orthogonally removed and the resulting carboxy group was functionalized with a glycine,thioester followed by standard trifluoroacetic acid (TFA) cleavage to produce the unprotected peptide,thioester. The structure of the synthetic protein was examined by far- and near-UV circular dichroism (CD), sedimentation equilibrium ultracentrifugation, and NMR and fluorescence spectroscopy. The spectroscopic methods show a highly helical and native-like monomeric protein consistent with the design. Heat-induced unfolding was studied by tryptophan absorbance and far-UV CD. The thermal unfolding of GTD-Lig occurs in two steps; a cooperative transition from the native state to an intermediate state and thereafter by noncooperative melting to the unfolded state. The intermediate exhibits the properties of a molten globule such as a retained native secondary structure and a compact hydrophobic core. The thermodynamics of GuHCl-induced unfolding were evaluated by far-UV CD monitoring and the unfolding exhibited a cooperative transition that is well-fitted by a two-state mechanism from the native to the unfolded state. GTD-Lig clearly shows the characteristics of a native protein with a well-defined structure and typical unfolding transitions. The design and synthesis presented herein is of general applicability for the construction of large monomeric proteins. [source] Folding Dynamics of 10-Residue ,-Hairpin Peptide ChignolinCHEMISTRY - AN ASIAN JOURNAL, Issue 5 2007Atsushi Suenaga Dr. Abstract Short peptides that fold into ,-hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7,,s) of the 10-residue ,-hairpin peptide chignolin, which is the smallest ,-hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen-bond network and the complete hydrophobic core as well as the arrangement of side-chain,side-chain interactions occur at approximately the same time. This three-step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the ,-hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa. [source] Study of Structural Stability of Cyclophilin A by NMR and Circular Dichroism SpectraCHINESE JOURNAL OF CHEMISTRY, Issue 7 2006Yan-Hong Shi Abstract The structural stability of cyclophilin A (CypA) was investigated using H/D exchange and temperature coefficients of chemical shifts of amide protons, monitored by 2D heteronuclear NMR spectroscopy. Amide proton exchange rates were measured by H/D exchange experiments for slow-exchange protons and measured by SEA (Solvent Exposed Amides)-HSQC experiments for fast-exchange protons. Temperature coefficients of chemical shifts and hydrogen exchange rates of amide protons show reasonably good correlation with the protein structure. Totally, 44 out of 153 non-proline assigned residues still exist in 86 d of hydrogen-deuterium exchange at 4 °C, suggesting that CypA structure should be highly stable. Residues in secondary structures of ,2, ,1, ,2, ,5, ,6 and ,7 might constitute the hydrophobic core of the protein. The change in free energy of unfolding (,Gu) of CypA was estimated to be (21.99±1.53) kJ·mol,1 by circular dichroism (CD). The large free energy change is also an indicator of the high structural stability. [source] Crystal structure of human coactosin-like protein at 1.9 Å resolutionPROTEIN SCIENCE, Issue 11 2004Xuemei Li Abstract Human coactosin-like protein (CLP) shares high homology with coactosin, a filamentous (F)-actin binding protein, and interacts with 5LO and F-actin. As a tumor antigen, CLP is overexpressed in tumor tissue cells or cell lines, and the encoded epitopes can be recognized by cellular and humoral immune systems. To gain a better understanding of its various functions and interactions with related proteins, the crystal structure of CLP expressed in Escherichia coli has been determined to 1.9 Å resolution. The structure features a central ,-sheet surrounded by helices, with two very tight hydrophobic cores on each side of the sheet. CLP belongs to the actin depolymerizing protein superfamily, and is similar to yeast cofilin and actophilin. Based on our structural analysis, we observed that CLP forms a polymer along the crystallographic b axis with the exact same repeat distance as F-actin. A model for the CLP polymer and F-actin binding has therefore been proposed. [source] | ||||||||||||||||||||||||||||