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PubMed1995

Human herpesvirus 6B origin-binding protein: DNA-binding domain and consensus binding sequence.

We previously demonstrated by a DNA-binding assay that the human herpesvirus 6B (HHV-6B) replication origin has a structure similar to those of alphaherpesviruses, although the HHV-6B and herpes simplex virus type 1 (HSV-1) origin-binding proteins (OBPs) and origins are not interchangeable. Here we describe additional properties of the interaction between HHV-6B OBP and the HHV-6B origin. Competitive electrophoretic mobility shift assays (EMSAs) with DNA duplexes containing single-base alterations allowed deduction of a consensus DNA sequence for HHV-6B-specific OBP binding, YGWYCWCCY, where Y is T or C and W is T or A, while that for HSV-1-specific binding was reported to be YGYTCGCACT. By EMSA, the HHV-6B OBP DNA-binding domain was mapped to a segment containing amino acids 482 to 770. However, in Southwestern (protein-DNA) blotting, the region sufficient for the DNA binding encompassed only amino acids 657 to 770. Similarly, Southwestern blotting showed that amino acids 689 to 851 of HSV-1 OBP had HSV-1 origin-binding activity, although this region was insufficient for origin binding in the EMSA. Although the longer DNA-binding domains identified by EMSA have marginal overall homology among HHV-6B and alphaherpesvirus OBP homologs, the smaller regions sufficient for the binding observed by Southwestern blotting have significant similarity. From these results, we propose a hypothesis that the DNA-binding domain of herpesvirus OBPs consists of two subdomains, one containing a conserved motif that contacts DNA directly, and another, less well conserved, that may modulate either the conformation or accessibility of the binding domain.

Amino Acid Sequence↗

PubMed1984

The stereochemistry of peptides containing alpha-aminoisobutyric acid.

The introduction of alpha-aminoisobutyric acid (Aib) into peptides dramatically limits the range of accessible backbone conformations. The presence of two geminal methyl groups at C alpha sterically compels Aib residues to largely favor structures in the right- or left-handed 3(10)/alpha-helical regions (phi approximately +/- 60 +/- 20 degrees, psi approximately +/- 30 +/- 20 degrees) of the peptide conformational map. Aib residues occur extensively in microbial peptides which form transmembrane channels. This observation has stimulated considerable interest in the stereochemistry of Aib peptides. This review summarizes theoretical studies on the conformations of Aib residues and examines the available data on solid-state structures, derived from single crystal X-ray diffraction studies. Crystal structures of over three dozen Aib-containing peptides, ranging in length from 2 to 11 residues, have been reported so far which exemplify various types of beta-turns, consecutive beta-turns, and helical structures. Examples of nonhydrogen bonded and cyclic structures are also described. The crystallographic results compare well with structural studies in solution, establishing that Aib peptides can provide rigid structural models for the development of spectroscopic methods of peptide conformational analysis.

Aminoisobutyric Acids↗

PubMed2000

Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster.

During the differentiation of naive Th cells into Th2 effector cells, the entire IL-4/IL-13 locus is remodeled into an accessible chromatin conformation. Here we show that ectopic expression and activation of Stat6 or GATA-3 in Th cells developing under Th1-polarizing conditions lead to the induction of chromatin remodeling not only at the flanking regions of the IL-4 and IL-13 genes but also at the IL-4/IL-13 intergenic regulatory region for the IL-4/IL-13/IL-5 gene cluster. Furthermore, we demonstrate that GATA-3 and another Th2-specific, inducible protein complex interact with the IL-4/IL-13 intergenic DNase I hypersensitive region specifically in Th2 cells.

Animals↗

PubMed1977

Circular dichroic and perturbation spectra of aromatic chromophores in rabbit tropomyosin. Topography of tyrosine residues.

1. Difference spectra of tryosyl residues obtained on denaturation of tropomycosin with urea or guanidinium chloride indicate that strong hydrophobic environments exist in the native coiled-coil state. 2. Solvent perturbation difference spectra indicate that tyrosyl residues are partially accessible to the solvent. The accessiblity decreases with increasing size of the solvent molecules. 3. Spectral pH titration of tyrosyl residues cannot provide information on the tyrosyl accessibility because conformational change accompanies the increase in pH. 4. Circular dichroism of tyrosyl and phenylalanyl residues is consistent with the effect of imposed conformational rigidity on the partially asymmetrical vibrational fine structure of the 1Lb absorption band of phenyl and benzyl chromophores; the effect is reduced by 70% on unfolding of tropomyosin.

Animals↗

PubMed1993

Human very low density lipoprotein structure: interaction of the C apolipoproteins with apolipoprotein B-100.

Very low density lipoproteins (VLDL) are a heterogenous population of particles differing in size and composition. Heparin-Sepharose chromatography yields three VLDL subfractions. Two subfractions, VLDLNR-1 and VLDLNR-2, which are not retained by heparin, contain little or no detectable apolipoprotein (apo)E. According to negative stain electron microscopy, VLDLNR-1 is slightly larger than VLDLNR-2. The third fraction, VLDLR, is composed of smaller particles that are retained by the heparin-Sepharose and contain apoE. The C apolipoproteins of the respective VLDL subfractions transfer to 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) single bilayer vesicles giving three subfractions designated VLDLNR-1-C, VLDLNR-2-C, and VLDLR-C. The protein, phospholipid, and cholesterol (free + esterified) contents decrease in the order VLDLR > VLDLNR-2 > VLDLNR-1. Triglyceride content decreases in the opposite order. POPC treatment of each VLDL subfraction increases the phospholipid and decreases the protein, triglyceride, and cholesteryl ester contents, while free cholesterol remains unchanged. According to immunological analysis of each subfraction with well-characterized monoclonal antibodies, the accessibility of some epitopes of apoB-100 on VLDL is changed by POPC treatment. Electron-microscopic analysis of POPC-treated VLDL subfraction reveals vacancies on the surfaces of each particle. VLDLNR-1, VLDLNR-2, and VLDLR are resistant to thrombin cleavage, whereas the lipoproteins lacking C apolipoproteins are not. Thrombin cleavage (8 h) of apoB-100 of VLDLNR-2-C and VLDLR-C gives two fragments, T1 and T2, that are converted to smaller fragments only after prolonged treatment. In contrast, apoB-100 of VLDLNR-1-C is converted into small fragments after 8 h thrombin treatment. These results suggest that removal of apoCs affects the accessibility and conformation of apoB-100 in the individual VLDL subfractions in the region near residue 3249, which is the primary thrombin cleavage site and the epitope of monoclonal antibody 4C11.

Apolipoprotein B-100↗

PubMed1994

Linker DNA accessibility in chromatin fibers of different conformations: a reevaluation.

New studies on chromatin fiber morphology, using the technique of scanning force microscopy (SFM), have caused us to reexamine recent analysis of nuclease digestion of chromatin. Chicken erythrocyte chromatin fibers, glutaraldehyde-fixed at 0, 10, and 80 mM NaCl, were imaged with the help of SFM. The chromatin fibers possessed a loose three-dimensional 30-nm structure even in the absence of added salt. This structure slightly condensed upon addition of 10 mM NaCl, and highly compacted, irregularly segmented fibers were observed at 80 mM NaCl. This sheds new light upon our previously reported analysis of the kinetics of digestion by soluble and membrane-immobilized micrococcal nuclease [Leuba, S. H., Zlatanova, J. & van Holde, K. (1994) J. Mol. Biol. 235, 871-880]. While the low-ionic-strength fibers were readily digested, the highly compacted structure formed at 80 mM NaCl was refractory to nuclease attack, implying that the linkers were fully accessible in the low-ionic-strength conformation but not in the condensed fibers. We now find that cleavage of the linker DNA by a small molecule, methidiumpropyl-EDTA-Fe(II), proceeds for all types of conformations at similar rates. Thus, steric hindrance is responsible for the lack of accessibility to micrococcal nuclease in the condensed fiber. Taken in total the data suggest that reexamination of existing models of chromatin conformation is warranted.

Animals↗

PubMed1998

Effect of ethanol on chromatin and nonhistone nuclear proteins in rat brain.

Changes in chromatin conformation and nonhistone nuclear protein composition were analyzed in various classes of nuclei from the brain of control and chronic ethanol fed rats. Conformational studies of chromatin by circular dichroism spectrophotometry showed an increased molar ellipticity [theta] of chromatin in neuronal, astrocyte and oligodendroglial nuclei due to ethanol treatment. The increased molar ellipticity directly indicates relaxed state of chromatin in these nuclei, which facilitates ready state of transcription and replication. Further, the circular dichroism spectrum, due to a change over point at approximately 260 nm also indicated the possibility of DNA-protein interactions governing chromatin conformation. In microglial nuclei, the circular dichroism spectrum showed a decrease in molar ellipticity due to ethanol treatment, indicating the existence of chromatin in a condensed state. This type of circular dichroism change points towards the possibility of closed conformation, which renders the gene sequences not accessible due to conformational constrains of the chromatin. Since circular dichroism changes indicated the involvement of DNA-protein interactions, changes in nonhistone nuclear proteins were analyzed in these classes of nuclei by two-dimensional gel electrophoresis. In astrocytes and oligodendrocytes two new proteins appeared in each type of nuclei while in neurons and microglial nuclei four different proteins were either completely missing or showed a decrease. These changes indicate the presence of dynamic flux of nonhistone nuclear proteins in chromatin. Taken together, the changes in chromatin conformation, associated with specific changes in non histone nuclear protein composition suggest the modulation of chromatin as a response to ethanol evoked stimulus and has relevance in the regulation of cellular responses to ethanol crisis in brain.

Alcoholism↗

PubMed2004

A novel method reveals that solvent water favors polyproline II over beta-strand conformation in peptides and unfolded proteins: conditional hydrophobic accessible surface area (CHASA).

In aqueous solution, the ensemble of conformations sampled by peptides and unfolded proteins is largely determined by their interaction with water. It has been a long-standing goal to capture these solute-water energetics accurately and efficiently in calculations. Historically, accessible surface area (ASA) has been used to estimate these energies, but this method breaks down when applied to amphipathic peptides and proteins. Here we introduce a novel method in which hydrophobic ASA is determined after first positioning water oxygens in hydrogen-bonded orientations proximate to all accessible peptide/protein backbone N and O atoms. This conditional hydrophobic accessible surface area is termed CHASA. The CHASA method was validated by predicting the polyproline-II (P(II)) and beta-strand conformational preferences of non-proline residues in the coil library (i.e., non-alpha-helix, non-beta-strand, non-beta-turn library derived from X-ray elucidated structures). Further, the method successfully rationalizes the previously unexplained solvation energies in polyalanyl peptides and compares favorably with published experimentally determined P(II) residue propensities. We dedicate this paper to Frederic M. Richards.

Hydrogen Bonding↗

PubMed1998

Conformationally variable Rab protein surface regions mapped by limited proteolysis and homology modelling.

Tryptic proteolysis of the small GTPases Rab4 and Rab5 is a multi-step, nucleotide-dependent process. Using N-terminal peptide sequencing, matrix-assisted laser desorption ionization-time-of-flight MS and molecular modelling, we identified the three initial sites of proteolysis in Rab5 as Arg-4, Arg-81 and Arg-197. Arg-4 and Arg-81 lie within regions previously implicated in Rab5 endocytic function, and Arg-197 lies in a region involved in membrane targeting. Topologically, Arg-81 lies within the conformationally variable Switch II region shown to be important for protein-protein interactions of other GTPases. Homology modelling studies on Rab5 indicate that the Arg-81 side chain is buried in the Rab5 GTP conformation, but is solvent-accessible in the GDP conformation, explaining the dependence of proteolysis on nucleotides. Peptide mapping of Rab4 was performed to take advantage of additional scissile bonds within Switch II to determine more precisely the limits of the nucleotide-dependent protease-accessible region. The Rab4 cleavage sites corresponded to Arg-81 and Pro-87 of Rab5, and taken together with the finding that Rab5 was not cleaved at Arg-91 this analysis defines an eight-residue surface-exposed conformationally variable region lying in the centre of Switch II. A sequence comparison of Rab proteins shows these eight residues to have a loosely conserved motif that we term Switch II(v) for its relative variability. C-terminal to Switch II(v) is a highly conserved Rab-specific YYRGA motif that we term Switch II(c) for its constant sequence. N-terminal to Switch II(v) is a sequence-invariant G-domain involved in nucleotide binding and hydrolysis. We propose that the Rab Switch II(v) region imparts specificity to nucleotide-dependent protein-protein interactions.

Amino Acid Sequence↗

PubMed2003

CADB: Conformation Angles DataBase of proteins.

Conformation Angles DataBase (CADB) provides an online resource to access data on conformation angles (both main-chain and side-chain) of protein structures in two data sets corresponding to 25% and 90% sequence identity between any two proteins, available in the Protein Data Bank. In addition, the database contains the necessary crystallographic parameters. The package has several flexible options and display facilities to visualize the main-chain and side-chain conformation angles for a particular amino acid residue. The package can also be used to study the interrelationship between the main-chain and side-chain conformation angles. A web based JAVA graphics interface has been deployed to display the user interested information on the client machine. The database is being updated at regular intervals and can be accessed over the World Wide Web interface at the following URL: http://144.16.71.148/cadb/.

Animals↗

PubMed1980

Anion transport across the red blood cell membrane and the conformation of the protein in Band 3.

Measuring the rate of dinitrophenylation of a specific lysine residue (called a) that is allosterically linked to the transfer site, it could be demonstrated that the anion transport protein may exist in two different conformational states, designated cis and trans. In the cis conformation a is easily accessible for reaction with dinitrofluorobenzene; in the trans conformation, a is less accessible. In the presence of the substrate anion Cl, the equilibrium between the cis and trans conformation is towards the cis conformation. Reversibly acting inhibitors of anion transport arrest the transport system, either predominantly in the cis or in the trans conformation. Phlorizin and certain positively charged derivatives of furosemide produce arrest in cis conformation, internal 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonate (APMB) and Ca++ in trans conformation. Within this frame of reference, the different susceptibilities of the transfer site to internal and external 4,4' diacetamido-2,2'-stilbene disulfonate (DAS) are interpreted on the assumption that the conformation of the transfer site changes during the transition of the transport protein from the cis to the trans conformation, so that in the trans conformation a reaction with DAS is no longer possible.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1983

The accessibility of the active site and conformation states of the beta 2 subunit of tryptophan synthase studied by fluorescence quenching.

The rate of quenching of the fluorescence of pyridoxal 5'-phosphate in the active site of the beta 2 subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide. The alpha subunit and the substrate L-serine substantially reduced the quenching rate. For iodide, the order of decreasing quenching was: Schiff's base of N alpha-acetyl-lysine with pyridoxal 5'-phosphate greater than holo beta 2 subunit greater than holo alpha 2 beta 2 complex approximately equal to holo beta 2 subunit + L-serine greater than holo alpha 2 beta 2 complex + L-serine. The coenzyme in the beta 2 subunit is apparently freely accessible to both iodide and acrylamide (kappa approximately equal to 2 X 10(9) M-1 s-1), but the alpha subunit and L-serine decrease the rate by factors of 2-5. Quenching of the fluorescence of the single tryptophan residue of the beta 2 subunit revealed that the apo and holo forms exist in different states, whereas the alpha subunit stabilizes a third conformation. As the alpha subunit binds to the beta 2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the beta 2 subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced. The alpha subunit strongly protects the active-site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein. Iodide induces dissociation of the holo alpha 2 beta 2 complex [E. W. Miles & M. Moriguchi (1977) J. Biol. Chem. 252, 6594-6599]. The effect of iodide on the fluorescence properties of holo alpha 2 beta 2 complex allows us to estimate an upper limit for the dissociation constant for the alpha 2 beta 2 complex of 10(-8) M, in the absence of iodide.

Acrylamides↗

PubMed2005

Ion-triggered multistate molecular switching device based on regioselective coordination-controlled ion binding.

Molecular devices capable of accessing different controlled conformational states, while optically signaling the occupied state, are attractive tools for nanotechnology since they relate to both areas of molecular mechanical devices and logic gates. We report here a simple molecular system that allows access to four distinct conformational and optical states. It is based on the regioselective complexation of metal ions to a heterocyclic ligand triad, which is dictated by the accessible coordination geometry and electrostatic properties of two distinct binding subunits. Thus, local conformational switching is brought about by tetrahedral coordination (of Cu(I)) or octahedral coordination (of M(2+) ions) to bidentate and tridentate binding subunits, respectively. The shape modifications undergone represent an ion-controlled nanomechanical device. They give controlled access to four different states that display different physico-chemical (e.g. optical) properties and provide a basis for logic gate operations.

Journal Article↗

PubMed1998

Arthritis-related B cell epitopes in collagen II are conformation-dependent and sterically privileged in accessible sites of cartilage collagen fibrils.

In collagen-induced arthritis, a murine autoimmune model for rheumatoid arthritis, immunization with native but not heat-denatured cartilage-specific collagen type II (CII) induces a B cell response that largely contributes to arthritogenicity. Previously, we have shown that monoclonal antibodies established from arthritis prone DBA/1 mice require the triple-helical conformation of their epitopes for antigen recognition. Here, we present a novel approach to characterize arthritis-related conformational epitopes by preparing a panel of 130 chimeric collagen X/CII molecules. The insertion of a series of CII cassettes into the triple-helical recombinant collagen X allowed for the first time the identification of five triple-helical immunodominant domains of 5-11 amino acid length, to which 75% of 36 monoclonal antibodies bound. A consensus motif, "R G hydrophobic," was found in all immunodominant epitopes. The antibodies were encoded by a certain combination of V-genes in germline configuration, indicating a role of the consensus motif in V-gene selection. The immunodominant domains are spread over the entire monomeric CII molecule with no apparent order; however, a highly organized arrangement became apparent when the CII molecules were displayed in the quarter-staggered assembly within a fibril. This discrete epitope organization most likely reflects structural constraints that restrict the exposure of CII epitopes on the surface of heterotypically assembled cartilage fibrils. Thus, our data suggest a preimmune B cell selection process that is biased by the accessibility of CII determinants in the intact cartilage tissue.

Animals↗

PubMed1998

Reversible peptide folding in solution by molecular dynamics simulation.

Long-standing questions on how peptides fold are addressed by the simulation at different temperatures of the reversible folding of a peptide in solution in atomic detail. Molecular dynamics simulations correctly predict the structure that is thermodynamically stable at 298 K, irrespective of the initial peptide conformation. The rate of folding and the free energy of folding at different temperatures are estimated. Although the conformational space potentially accessible to the peptide is extremely large, very few conformers (10(1) to 10(2)) are significantly populated at 20 K above the melting temperature. This implies that the search problem in peptide (or even protein) folding is surmountable using dynamics simulations.

Computer Simulation↗

PubMed2000

Substrate specificity and signal transduction pathways in the glucose-specific enzyme II (EII(Glc)) component of the Escherichia coli phosphotransferase system.

Escherichia coli adapted to glucose-limited chemostats contained mutations in ptsG resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EII(Glc)) and resulting in increased transport of glucose and methyl-alpha-glucoside. The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner. By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R). The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol. The mutations belonged to two classes. First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly. More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increased ptsG transcription in the absence of glucose. Increased expression of wild-type EII(Glc) was not by itself sufficient for growth with other sugars. A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EII(Glc). The conformation of EII(Glc) elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of pts genes.

Amino Acid Substitution↗

PubMed1988

Ligand activation causes a phosphorylation-dependent change in platelet-derived growth factor receptor conformation.

The effect of ligand binding on platelet-derived growth factor (PDGF) receptor conformation was examined using peptide antibodies directed against specific receptor domains. Antiserum 83, which was directed to the receptor's carboxyl terminus (residues 934-951), preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the antibody to the carboxyl terminus. The activated receptor conformation was induced by three different forms of PDGF (AA and BB homodimers and AB heterodimers) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor, sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by adenyl-5'-yl imidodiphosphate, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in Chinese hamster ovary fibroblasts transfected with wild-type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis even though 125I-PDGF binding was normal. These findings show that ligand binding elicits a phosphorylation-dependent change in PDGF receptor conformation that may be important for receptor function.

Adenylyl Imidodiphosphate↗

PubMed2005

Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases.

Flavoprotein monooxygenases are involved in a wide variety of biological processes including drug detoxification, biodegradation of aromatic compounds in the environment, biosynthesis of antibiotics and siderophores, and many others. The reactions use NAD(P)H and O2 as co-substrates and insert one atom of oxygen into the substrate. The flavin-dependent monooxygenases utilize a general cycle in which NAD(P)H reduces the flavin, and the reduced flavin reacts with O2 to form a C4a-(hydro)peroxyflavin intermediate, which is the oxygenating agent. This complicated catalytic process has diverse requirements that are difficult to be satisfied by a single site. Two general strategies have evolved to satisfy these requirements. para-Hydroxybenzoate hydroxylase, the paradigm for the single-component flavoprotein monooxygenases, is one of the most thoroughly studied of all enzymes. This enzyme undergoes significant protein and flavin dynamics during catalysis. There is an open conformation that gives access of substrate and product to solvent, and a closed or in conformation for the reaction with oxygen and the hydroxylation to occur. This closed form prevents solvent from destabilizing the hydroperoxyflavin intermediate. Finally, there is an out conformation achieved by movement of the isoalloxazine toward the solvent, which exposes its N5 for hydride delivery from NAD(P)H. The protein coordinates these dynamic events during catalysis. The second strategy uses a reductase to catalyze the reduction of the flavin and an oxygenase that uses the reduced flavin as a substrate to react with oxygen and hydroxylate the organic substrate. These two-component systems must be able to transfer reduced flavin from the reductase to the oxygenase and stabilize a C4a-peroxyflavin until a substrate binds to be hydroxylated, all before flavin oxidation and release of H2O2. Again, protein dynamics are important for catalytic success.

4-Hydroxybenzoate-3-Monooxygenase↗