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PubMed2003

The luteinizing hormone receptor: influence of buffer composition on ligand binding and signaling of wild type and mutant receptors.

There is evidence that ligand binding to and ligand-mediated signaling by the luteinizing hormone receptor (LHR) are influenced by buffer conditions, including ionic type and strength, an issue that becomes important in comparing functional parameters obtained on receptor mutants under different conditions. In order to study this phenomenon, we performed binding (kinetic and saturation) and signaling studies of human chorionic gonadotropin (hCG) with wild type (wt) LHR and several mutants expressed in COS-7 cells using two common buffer systems. One buffer was of low ionic strength and contained a low concentration of Na+, while the other had a near-physiological concentration of Na+. Emphasis was placed on mutations of two amino acid residues in the hinge region of the ectodomain (E332 and D333). It was found that the buffer of higher ionic strength, primarily from Na+, led to an increase of about 4-fold in the Kd of hCG binding to wt and mutant LHRs. The reduced binding affinities were attributable to a comparable reduction in the rate constants of association, with no significant differences in the calculated rate constants of dissociation in the two buffers. Analysis of the signaling properties of these mutants showed that, when corrected for the amount of hCG bound under the conditions of the signaling assay, the maximal ligand-mediated cAMP produced in cells maintained in the buffer of low ionic strength was comparable for wt LHR and the mutants, only the D333A mutant being somewhat elevated. In the buffer of higher ionic strength, however, the response by wt LHR was significantly greater than that of the mutants. These results show that E332 and D333 are important in hormone-mediated signaling, but only in the buffer of higher Na+ concentration. In addition to mutants of these two residues, the buffer of higher ionic strength also led to reduced binding to a number of mutants throughout the receptor. Since these mutants included additional replacements in the ectodomain and transmembrane helices 6 and 7, the general nature of the buffer effect on wt and mutant LHRs suggests that electrostatic effects are contributing to ligand binding and/or that the LHR ectodomain may exist in two conformations, one being more accessible to ligand at reduced ionic strength.

Amino Acid Sequence↗
PubMed2000

Peptide display on bacterial flagella: principles and applications.

Expression of foreign peptides as fusions to bacterial cell surface proteins has gained increasing attention in basic, as well as applied research during the last decade. A wide range of heterologous peptides have been expressed, and the spectrum of available carrier proteins is also wide. The choice of carrier protein is frequently ruled by the application of the fusion protein constructed. This review is focused on flagella display, which is based on genetic fusion of foreign peptides into a surface-exposed, dispensable region of flagellin, the flagellar major subunit present in thousands of copies per filament. Expression of these constructs in flagellin-deficient host strains results in hybrid flagella carrying the heterologous peptides in thousands of intimately-associated copies. The first and still most frequent application of flagella display is the construction of novel recombinant vaccines. Flagella display has also been used in peptide display as an alternative to the phage-display technique. One application involves fusion into a disulfide loop of Escherichia coli thioredoxin that has been inserted into flagellin, this system facilitates expression of random peptides in a conformationally constrained manner readily accessible on the flagellar surface. The random peptide library has been applied in antibody epitope mapping and is suitable for biopanning procedures in the study of ligand-receptor interactions. Many bacterial adhesins are of complex nature and thereby difficult to analyse by conventional methods. Direct flagella display has proven to be applicable also in bacterial adhesion technology since large fragments, up to 302 amino acid residues in length, of bacterial adhesins can be functionally expressed as fusions to flagellin. Hybrid flagella are easily purified and can easily be analysed for binding to various targets, such as immobilized proteins, tissue sections, as well as cell cultures.

Adhesins, Bacterial↗
PubMed2001

Design of multistable RNA molecules.

We show that the problem of designing RNA sequences that can fold into multiple stable secondary structures can be transformed into a combinatorial optimization problem that can be solved by means of simple heuristics. Hence it is feasible to design RNA switches with prescribed structural alternatives. We discuss the theoretical background and present an efficient tool that allows the design of various types of switches. We argue that both the general properties of the sequence structure map of RNA secondary structures and the ease with which our design tool finds bistable RNAs strongly indicates that RNA switches are easily accessible in evolution. Thus conformational switches are yet another function for which RNA can be employed.

Base Pairing↗
PubMed1988

Deoxyribonuclease I sensitivity of the T-DNA ipt gene is associated with gene expression.

We have analyzed the chromatin structure of the T-DNA isopentenyl transferase gene, ipt, in four Nicotiana tabacum crown gall tumor lines. These four transformed lines contain identical T-DNA inserts and are derivatives of a single clone that did not exhibit any tumorous properties and contained a highly methylated, nonexpressed copy of T-DNA. One of the derivatives also does not exhibit tumorous properties, and the T-DNA of this line is not expressed. The other three lines have reverted to tumorous growth either spontaneously or after treatment with the inhibitor of DNA methylation, 5-azacytidine. Concomitant with this reversion to tumorous growth, expression of the ipt gene of these lines has reinitiated. In the lines that express the ipt gene, the chromatin structure of this gene exists in a conformation that is more accessible to DNase I than in the line in which this gene is not expressed. The level of ipt expression and DNase I sensitivity was independent of the process by which the transformed cell lines reverted to tumorous growth. The relationship of chromatin structure to gene expression and DNA methylation in these lines is discussed.

Chromatin↗
PubMed1989

Orientation of the N-terminal region of the membrane-bound ADP/ATP carrier protein explored by antipeptide antibodies and an arginine-specific endoprotease. Evidence that the accessibility of the N-terminal residues depends on the conformational state of the carrier.

Two peptides corresponding to the amino acid sequences 1-11 (N-terminal peptide) and 288-297 (C-terminal peptide) of beef heart ADP/ATP carrier have been synthesized. After coupling to ovalbumin, they were injected into rabbits to raise polyclonal antibodies. The specificities of the generated antibodies were tested by enzyme-linked immunosorbent assay (ELISA) and (or) Western blot. Anti-N-terminal antibodies and anti-C-terminal antibodies reacted specifically with the corresponding peptide. However, only anti-N-terminal antibodies reacted with the isolated ADP/ATP carrier; they also reacted with the membrane-bound carrier in freeze-thawed mitochondria and mitoplasts, indicating that the first 10 amino acid residues of the membrane-bound carrier in mitochondria face the cytosol. On the basis that the ADP/ATP carrier can adopt two conformations, one trapped by carboxyatractyloside (CATR conformation) and the other by bongkrekic acid (BA conformation), the reactivity of the anti-N-terminal antibodies to the ADP/ATP carrier in mitoplasts or freeze-thawed mitochondria was tested for each conformation of the carrier. Only in the CATR conformation was the N-terminal region of the membrane-bound carrier reactive to the N-terminal antibodies; the contrasting weak reactivity of the carrier in the BA conformation suggested that the transition from the CATR conformation to the BA conformation results in a restricted conformation of the peptide chain corresponding to the first 10 amino acid residues or its partial burying in the lipid bilayer. These immunological data were complemented by enzymatic data pertaining to proteolysis of the membrane-bound ADP/ATP carrier by an arginine-specific endoprotease.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Local and global effects of metal binding within the small subunit of ribonucleotide reductase.

Each beta-protomer of the small betabeta subunit of Escherichia coli ribonucleotide reductase (R2) contains a binuclear iron cluster with inequivalent binding sites: Fe(A) and Fe(B). In anaerobic Fe(II) titrations of apoprotein under standard buffer conditions, we show that the majority of the protein binds only one Fe(II) atom per betabeta subunit. Additional iron occupation can be achieved upon exposure to O2 or in high glycerol buffers. The differential binding affinity of the A- and B-sites allows us to produce heterobinuclear Mn(II)Fe(II) and novel Mn(III)Fe(III) clusters within a single beta-protomer of R2. The oxidized species are produced with H2O2 addition. We demonstrate that no significant exchange of metal occurs between the A- and B-sites, and thus the binding of the first metal is under kinetic control, as has been suggested previously. The binding of first Fe(II) atom to the active site in a beta-protomer (betaI) induces a global protein conformational change that inhibits access of metal to the active site in the other beta-protomer (betaII). The binding of the same Fe(II) atom also induces a local effect at the active site in betaI-protomer, which lowers the affinity for metal in the A-site. The mixed metal FeMn species are quantitatively characterized with electron paramagnetic resonance spectroscopy. The previously reported catalase activity of Mn2(II)R2 is shown not to be associated with Mn.

Apoproteins↗
PubMed2005

Differential activation of a latent polyphenol oxidase mediated by sodium dodecyl sulfate.

A kinetic study of the activity of soluble and membrane-bound latent polyphenol oxidase (PPO) extracted from beet root (Beta vulgaris) was carried out. For the first time, two types of behavior (hyperbolic and sigmoid) are reported in the same enzyme for PPO activation by the surfactant sodium dodecyl sulfate (SDS), depending on substrate nature. A kinetic model based on cooperative systems is developed to describe the activation effect of SDS, enabling the determination of the number of surfactant molecules binding to the enzyme in the activation process. The results indicate that the active site of the enzyme is not affected by SDS and that a stepwise conformational change favors the access of hydrophobic substrates compared to hydrophilic ones. Differential activation of PPO mediated by SDS may be of relevance in the control of PPO activity since the enzyme is able to express activity toward a specific substrate while remaining latent to others.

Beta vulgaris↗
PubMed1999

Three-dimensional quantitative structure-activity relationship study on cyclic urea derivatives as HIV-1 protease inhibitors: application of comparative molecular field analysis.

Three-dimensional quantitative structure-activity relationship (3D-QSAR) models have been developed using comparative molecular field analysis (CoMFA) on a large data set (118 compounds) of diverse cyclic urea derivatives as protease inhibitors against the human immunodeficiency virus type 1 (HIV-1). X-ray crystal structures of HIV-1 protease bound with this class of inhibitors were used to derive the most probable bioactive conformations of the inhibitors. The enzyme active site was used as a constraint to limit the number of possible conformations that are sterically accessible. The test sets have been created keeping in mind structural diversity as well as the uniform simple statistical criteria (mean, standard deviation, high and low values) of the protease inhibitory activities of the molecules compared to the training sets. Multiple predictive models have been developed with the training sets (93 compounds in each set) and validated with the corresponding test sets (25 compounds in each set). All the models yielded high predictive correlation coefficients (q2 from 0.699 to 0.727), substantially high fitted correlation coefficients (r2 from 0.965 to 0.973), and reasonably low standard errors of estimates (S from 0. 239 to 0.265). The steric and electrostatic effects have approximately equal contributions, 45% and 55% (approximately), respectively, toward explaining protease inhibitory activities. This analysis yielded models with significant information on steric and electrostatic interactions clearly discerned by the respective coefficient contour plots when overlapped on the X-ray structure of the HIV-1 protease. The HINT CoMFA study revealed significant contribution of hydrophobicity toward protease inhibitory activity. The 3D visualization technique utilizing these contour plots as well as the receptor site geometry may significantly improve our understanding of the inhibitor-protease (HIV-1) interactions and help in designing compounds with improved activity.

Anti-HIV Agents↗
PubMed2003

Short and diverse route toward complex natural product-like macrocycles.

[reaction: see text] A general strategy toward macrocyclic compounds using multicomponent reaction (MCR) chemistry, e.g., Passerini and Ugi variants, and ring-closing metathesis (RCM) is introduced. The corresponding bifunctional isocyanides carboxylic acids bearing a terminal olefin are easy to prepare from the corresponding commercially available starting materials. Advantageously, this strategy allows fast access to a diverse conformational space of natural product-like macrocycles and could thus be of interest in the discovery of novel bioactive agents.

Alkenes↗
PubMed1997

Decreased susceptibility to calpains of v-FosFBR but not of v-FosFBJ or v-JunASV17 retroviral proteins compared with their cellular counterparts.

The c-Fos and c-Jun transcription factors are rapidly turned over in vivo. One of the multiple pathways responsible for their breakdown is probably initiated by calpains, which are cytoplasmic calcium-dependent cysteine proteases. The c-fos gene has been transduced by two murine oncogenic retroviruses called Finkel-Biskis-Jenkins murine sarcoma virus (FBJ-MSV) and Finkel-Biskis-Reilly murine sarcoma virus (FBR-MSV); c-jun has been transduced by the chicken avian sarcoma virus 17 (ASV17) retrovirus. Using an in vitro degradation assay, we show that the mutated v-FosFBR, but not v-FosFBJ or v-JunASV17, is resistant to calpains. This property raises the interesting possibility that decreased sensitivity to calpains might contribute to the tumorigenic potential of FBR-MSV by allowing greater accumulation of the protein that it encodes in infected cells. It has also been demonstrated that resistance to cleavage by calpains does not result from mutations that have accumulated in the Fos moiety of the viral protein but rather from the addition of atypical peptide motifs at its both ends. This observation raises the interesting possibility that homologous regions in viral and cellular Fos either display slightly different conformations or are differentially accessible to interacting proteins.

Avian Sarcoma Viruses↗
PubMed1998

Protected-site phosphorylation of protein kinase C in hippocampal long-term potentiation.

One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the alpha and betaII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a "protected site" model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.

Amino Acid Sequence↗
PubMed2002

Crystal structure of saposin B reveals a dimeric shell for lipid binding.

Saposin B is a small, nonenzymatic glycosphingolipid activator protein required for the breakdown of cerebroside sulfates (sulfatides) within the lysosome. The protein can extract target lipids from membranes, forming soluble protein-lipid complexes that are recognized by arylsulfatase A. The crystal structure of human saposin B reveals an unusual shell-like dimer consisting of a monolayer of alpha-helices enclosing a large hydrophobic cavity. Although the secondary structure of saposin B is similar to that of the known monomeric members of the saposin-like superfamily, the helices are repacked into a different tertiary arrangement to form the homodimer. A comparison of the two forms of the saposin B dimer suggests that extraction of target lipids from membranes involves a conformational change that facilitates access to the inner cavity.

Amino Acid Sequence↗
PubMed1996

Interactions of protein antigens with antibodies.

There are now several crystal structures of antibody Fab fragments complexed to their protein antigens. These include Fab complexes with lysozyme, two Fab complexes with influenza virus neuraminidase, and three Fab complexes with their anti-idiotype Fabs. The pattern of binding that emerges is similar to that found with other protein-protein interactions, with good shape complementarity between the interacting surfaces and reasonable juxtapositions of polar residues so as to permit hydrogen-bond formation. Water molecules have been observed in cavities within the interface and on the periphery, where they often form bridging hydrogen bonds between antibody and antigen. For the most part the antigen is bound in the middle of the antibody combining site with most of the six complementarity-determining residues involved in binding. For the most studied antigen, lysozyme, the epitopes for four antibodies occupy approximately 45% of the accessible surface area. Some conformational changes have been observed to accompany binding in both the antibody and the antigen, although most of the information on conformational change in the latter comes from studies of complexes with small antigens.

Animals↗
PubMed1995

The effect of mersalyl on inositol trisphosphate receptor binding and ion channel function.

A number of thiol-reactive agents induce repetitive Ca2+ spiking in cells by a mechanism thought to involve sensitization of the inositol 1,4,5-trisphosphate receptor (IP3R). To further define the basis of this interaction, we have studied the effect of several thiol-reactive agents on [3H]IP3 binding, IP3-gated channel activity, and conformation of the IP3R in membranes from hepatocytes, cultured WB rat liver epithelial cells, and cerebellum microsomes. At 4 degrees C, the organomercurial thiol-reactive agent mersalyl markedly stimulates (3-4fold) [3H]IP3 binding to permeabilized hepatocytes. The closely related molecule, thimerosal, has only a small stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of mersalyl was associated with a decrease in Kd of the IP3R with no change in Bmax. Mersalyl was without effect on detergent-solubilized hepatocyte binding sites or on the [3H]IP3 binding activity of cerebellum microsomes. In contrast to thimerosal, which potentiates IP3-mediated Ca2+ release, mersalyl blocked IP3-gated Ca2+ channels. Mersalyl pretreatment of WB membranes altered the pattern of immunoreactive receptor fragments generated upon subsequent cleavage of the receptor with proteinase K. This effect was not reproduced by thimerosal and was also not observed in experiments on cerebellum microsomes. We conclude that the WB cell and brain IP3 receptors are differently regulated by modification of thiol groups. Reaction of the WB cell IP3 receptor with mersalyl alters its conformation and modifies the accessibility of sites on the protein that are cleaved by proteinase K. In the presence of mersalyl, the receptor has high affinity for IP3 but is inactive as a Ca2+ channel. This contrasts with the high affinity receptor/active Ca2+ channel induced by thimerosal, suggesting that even closely related thiol agents may interact at different thiol groups.

Animals↗
PubMed2004

Oxidative folding of Amaranthus alpha-amylase inhibitor: disulfide bond formation and conformational folding.

Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus alpha-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process.

Amaranthus↗
PubMed2005

Presenilin-1 D257A and D385A mutants fail to cleave Notch in their endoproteolyzed forms, but only presenilin-1 D385A mutant can restore its gamma-secretase activity with the compensatory overexpression of normal C-terminal fragment.

The enzyme gamma-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the gamma-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other gamma-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of gamma-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their gamma-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349-467). However, the defective PS-1 D257A mutant could not restore their gamma-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the gamma-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.

Amyloid Precursor Protein Secretases↗
PubMed2006

5-methyltetrahydrofolate is bound in intersubunit areas of rat liver folate-binding protein glycine N-methyltransferase.

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.

Animals↗
PubMed1993

Differential post-irradiation caffeine response in normal diploid versus SV40-transformed human fibroblasts: potential role of nuclear organization and protein-composition.

To test the hypothesis that the enhancement of cell killing by post-irradiation treatment with caffeine (CAF) is mediated by alterations in chromatin structure, several nuclear parameters were examined in both caffeine-responsive and non-responsive cell lines. Cell killing, as determined by clonogenic assay, was not enhanced by post-irradiation treatment with 5 mM caffeine in a human diploid fibroblast line (AG1522) but an effect was seen in a SV40 T-antigen transformed derivative (1522-a). CAF caused a complete reversal of the radiation-induced G2 + S phase cell-cycle delays in the transformed cell line but only a partial reversal was noted for the parental cell line. The nuclear endpoints examined, which may be indicative of chromatin conformational changes, included enzymatic accessibility, DNA loop structure, and nuclear protein composition. In assays of the ability of DNA to undergo supercoiling changes, it was found that nucleoids isolated from CAF-treated cells had a significantly reduced propidium-iodide relaxable DNA loop size. The constraints to DNA unwinding produced by CAF were also maintained even in the presence of large numbers of single strand breaks produced by a test dose of radiation (10 Gy). This effect did not correlate well with the ability of CAF to enhance radiation-induced cell killing. The two other nuclear endpoints did detect differences between the normal and transformed cell lines. CAF had no effect on the DNase I digestion kinetics of the normal fibroblasts. However, in the transformed cell line, CAF appeared to render an additional 10-15% of the genome accessible to DNase I digestion. Several radiation and CAF-induced changes in the polypeptide pattern of isolated nucleoids were detected after metabolic labelling with 35S-methionine or 32P-orthophosphoric acid. While the identities of these proteins remain to be established, many had relative molecular weights similar to the other reported radiation-altered proteins and human cell cycle control gene products. The present cell lines should provide a convenient system in which to identify a nuclear protein change specifically associated with the ability of CAF to enhance radiation-induced cell killing.

Caffeine↗