Showing papers on "Conformational change published in 1995"
TL;DR: X-ray crystal structures show that the substituted residues complement regions of the protein surface that are used for substrate recognition in the native enzyme that support a relationship between stability and function for T4 lysozyme.
Abstract: Enzymes are thought to use their ordered structures to facilitate catalysis. A corollary of this theory suggests that enzyme residues involved in function are not optimized for stability. We tested this hypothesis by mutating functionally important residues in the active site of T4 lysozyme. Six mutations at two catalytic residues, Glu-11 and Asp-20, abolished or reduced enzymatic activity but increased thermal stability by 0.7-1.7 kcal.mol-1. Nine mutations at two substrate-binding residues, Ser-117 and Asn-132, increased stability by 1.2-2.0 kcal.mol-1, again at the cost of reduced activity. X-ray crystal structures show that the substituted residues complement regions of the protein surface that are used for substrate recognition in the native enzyme. In two of these structures the enzyme undergoes a general conformational change, similar to that seen in an enzyme-product complex. These results support a relationship between stability and function for T4 lysozyme. Other evidence suggests that the relationship is general.
680 citations
TL;DR: Examination of the biophysical properties of the mutant Hsp70s reveals a change in the overall shape and conformation of the protein consistent with reduced interactions between the two domains, which suggest that the EEVD motif is involved in the intramolecular regulation of HSp70 function and intermolecular interactions with HDJ‐1.
Abstract: The Hsp70 family of molecular chaperones has an essential role in the synthesis, folding and translocation of the nascent peptide chain. While the general features of these activities are well documented, less is understood about the regulation of these activities. The ATPase rate is stimulated by non-native proteins, furthermore, interaction with ATP leads to the release of protein substrate concurrent with a conformational change in Hsp70. One interpretation of these data is that the two domains of Hsp70 interact. In the process of mapping the carboxyl-terminal boundary of the substrate binding domain for human Hsp70, we identified a regulatory motif, EEVD, which is conserved at the extreme carboxyl terminus among nearly all cloned cytosolic eukaryotic Hsp70s. Deletion or mutation of EEVD affects the ATPase activity, the ability to interact with substrates, and interferes with the ability of the mutant Hsp70 to interact with HDJ-1 in the refolding of denatured firefly luciferase. Examination of the biophysical properties of the mutant Hsp70s reveals a change in the overall shape and conformation of the protein consistent with reduced interactions between the two domains. These data suggest that the EEVD motif is involved in the intramolecular regulation of Hsp70 function and intermolecular interactions with HDJ-1.
427 citations
TL;DR: P21ras is essential for NO-induced downstream signaling, such as NF-κB activation, and that endogenous NO can activate p21ras in the same cell, and it is suggested that NO activates p21ra by an action which mimics that of guanine nucleotide exchange factors.
364 citations
TL;DR: Peptide penetration into the lipid membrane and peptide aggregation at the membrane surface are proposed as possible mechanisms to explain the lipid-induced random coil<-->beta-structure transition.
318 citations
TL;DR: The data provide the first direct evidence for ligand-specific conformational changes occurring in a G protein-coupled receptor in response to drug binding, and demonstrate the potential of fluorescence spectroscopy as a tool for further delineating the molecular mechanisms of drug action at G Protein-Coupled receptors.
267 citations
TL;DR: The additive effects of mutations at this locus and at a conserved helix 7 locus investigated in the 5-HT2A receptor suggest that these residues are adjacent in space and interact and implicate both loci in a common hydrogen-bonding network underlying receptor activation by agonist.
212 citations
TL;DR: One-dimensional 1H and two-dimensional 15N-1H shift correlation NMR spectra of myristoylated recoverin measured as a function of Ca2- concentration show that a concerted conformational change occurs when two Ca2+ are bound.
193 citations
TL;DR: A general mechanism for guanine nucleotide discrimination, in common with that observed in high affinity GTP-binding proteins, involving the formation of a pair of hydrogen bonds between the aspartic acid side chain and N1 and N2 of the Guanine ring is proposed.
189 citations
TL;DR: The essential dynamics method was used to study differences in dynamics between the apo and holo forms of CRBP, and showed inhibition of essential motions upon ligand binding, and revealed large correlated motions of Retinol with regions of the protein, pointing to a possible retinol entry/exit site.
Abstract: The cellular retinol-binding protein (CRBP) is an intracellular retinol carrier protein belonging to a family of hydrophobic ligand-binding proteins, It transports retinol to specific locations in the cell where, for instance, it is esterified for storage, Recently solved crystallographic structures of CRBP homologues with and without bound ligand do not provide evidence for a ligand-induced conformational change, However, it has been shown that there is a difference in binding of holo-CRBP and apo-CRBP to lecithin-retinol acyltransferase, Moreover, proteolysis of holo-CRBP and apo-CRBP yields different products, indicating a difference in structure or dynamics between the two forms, Here, we present the results of molecular dynamics simulations of holo-CRBP and apo-CRBP, The simulations show a significant difference in conformation, in agreement with experimental results, The essential dynamics method was used to study differences in dynamics between the apo and hole forms of CRBP, and showed inhibition of essential motions upon ligand binding, It also revealed large correlated motions of retinol with regions of the protein, pointing to a possible retinol entry/exit site.
168 citations
TL;DR: It is shown that CyaA exposed to CaCl2 could retain membrane binding capability and hemolytic activity when it was further assayed in the presence of an excess of EGTA, and circular dichroism spectroscopy analysis showed that calcium binding to the low affinity sites induces a large conformational change of CyA, as revealed by an important increase in the content of α-helical structures.
161 citations
TL;DR: The gating mechanism in olfactory and rod channels expressed in Xenopus oocytes was studied and Ni2+ exerted its effect by binding preferentially to the closed configuration of the channel, thereby destabilizing the opening conformational change.
TL;DR: The change in the secondary structure of T4 lysozyme upon adsorption to silica particles was studied with circular dichroism and the rate of conformational change differed among the three proteins and was fastest for the tryptophan mutant.
TL;DR: The role of the individual subunits of the tetrameric channel in the C-type inactivation conformational change is investigated by comparing the inactivation rates of channels constructed from different combinations of subunits, and the relationship between the in activation rate and the number of fast subunits is exponential.
TL;DR: In this paper, single point mutations in the ATP binding site of hamster BiP, isolated recombinant proteins, and characterized them in terms of their affinity for ATP and ADP, their ability to undergo a conformational change upon nucleotide binding, and their rate of ATP hydrolysis.
TL;DR: Limited proteolysis of in vitro produced human androgen receptor was used to probe the different conformations of the receptor after binding of androgens and several antiandrogens, implying that, after the initial rapid binding of ligand, androgens induce a conformational change of the receptors, a process that also involves release of associated proteins.
TL;DR: The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF‐Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.
Abstract: The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood. We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP [3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench-flow technique. We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate. In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen. The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.
TL;DR: The use of 2-AP is described as a direct spectroscopic probe of the mechanism of nucleotide incorporation by Escherichia coli Pol I Klenow fragment (KF) and bacteriophage T4 DNA polymerase and the existence of a similar nonchemical step in the mechanism that features hydrogen bonding between the incoming and template bases is demonstrated.
Abstract: The fluorescent properties and their sensitivity to the surrounding environment of the nucleotide analog 2-aminopurine (2-AP) have been well documented. In this paper we describe the use of 2-AP as a direct spectroscopic probe of the mechanism of nucleotide incorporation by Escherichia coli Pol I Klenow fragment (KF) and bacteriophage T4 DNA polymerase. The nucleotidyl transfer reaction may be monitored in real time by following the fluorescence of 2-AP, allowing the detection of transient intermediates along the reaction pathway that are inaccessible through traditional radioactive assays. Previous studies with Klenow fragment [Kuchta, R. D., Mizrahi, V., Benkovic, P. A., Johnson, K. A., & Benkovic, S. J. (1987) Biochemistry 26, 8410-8417] have revealed the presence of a nonchemical step prior to chemistry and have identified this conformational change as the rate-limiting step of correct nucleotide incorporation. During correct incorporation, phosphodiester bond formation occurs at a rate greater than the conformational change and has not been measured. However, during misinsertion, the rate of the chemical step becomes partially rate limiting and it becomes possible to detect both steps. We have successfully decoupled the chemical and conformational change steps for nucleotide insertion by KF using the misincorporation reaction, and we present direct spectroscopic evidence for an activated KF'-DNA-dNTP species following the conformational change step which features hydrogen bonding between the incoming and template bases. In addition, we have utilized these same experiments to demonstrate the existence of a similar nonchemical step in the mechanism of dNTP incorporation by bacteriophage T4 DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The luminal domain of calnexin is identified as responsible for binding substrates, Ca, and Mg-ATP, and the association in vivo with a coexpressed secretory glycoprotein substrate, human immunodeficiency virus type I gp120 is revealed.
TL;DR: The conformations of nitrogen-in-the-ring sugars and their N-alkyl derivatives were studied from 1H NMR analyses, mainly using 3J(H,H) coupling constants and quantitative NOE experiments, and it was shown that the C6 OH group in 1 is predominantly equatorial to the piperidine ring, while that in 2 or 3 is predominantly axial, and its N-alksyl group is oriented equatorially.
Abstract: The conformations of nitrogen-in-the-ring sugars and their N-alkyl derivatives were studied from 1H NMR analyses, mainly using 3J(H,H) coupling constants and quantitative NOE experiments. No significant difference was seen in the ring conformation of 1-deoxynojirimycin (1), N-methyl-1-deoxynojirimycin (2), and N-butyl-1-deoxynojirimycin (3). However, it was shown that the C6 OH group in 1 is predominantly equatorial to the piperidine ring, while that in 2 or 3 is predominantly axial, and its N-alkyl group is oriented equatorially. In the furanose analogues 1,4-dideoxy-1,4-imino-D-arabinitol (4) and its N-methyl (5) and N-butyl (6) derivatives, the five-membered ring conformation differed significantly by the presence or absence of the N-substituted group and the length of the N-alkyl chain. Compound 3 reduced its inhibitory effect on almost all glycosidases, resulting in an extremely specific inhibitor for processing alpha-glucosidase I since N-alkylation of 1 is known to enhance both the potency and specificity of this enzyme in vitro and in vivo. This preferred (C6 OH axial) conformation in 2 and 3 appears to be responsible for their strong alpha-glucosidase I activity. Compound 4 is a good inhibitor of intestinal alpha-glucohydrolases, alpha-glucosidase II, and Golgi alpha-mannosidases I and II, but its N-alkyl derivatives 5 and 6 markedly decreased inhibitory potential for all enzymes tested. In the case of 2,5-dideoxy-2,5-imino-D-mannitol (DMDP, 7), which is a potent beta-galactosidase inhibitor, its N-methyl (8) and N-butyl (9) derivatives completely lost potency toward beta-galactosidase as well. N-Alkylation of compounds 4 and 7, known well as potent yeast alpha-glucosidase inhibitors, resulted in a serious loss of inhibitory activity toward yeast alpha-glucohydrolases. Activity of these nine analogues against HIV-1 replication was determined, based on the inhibition of virus-induced cytopathogenicity in MT-4 and MOLT-4 cells. Compounds 2 and 3, which are better inhibitors of alpha-glucosidase I than 1, proved active with EC50 values of 69 and 49 micrograms/mL in MT-4 cells and 100 and 37 micrograms/mL in MOLT-4 cells, respectively, while none of the furanose analogues exhibited any inhibitory effects on HIV-1. The change in potency and specificity of bioactivity by N-alkylation of nitrogen-in-the-ring sugars appears to be correlated with their conformational change.
TL;DR: The topology and post-translational modifications of microsomal 11β-hydroxysteroid dehydrogenase (11βDH) was investigated using the approaches of protein structure analysis, finding an absence of a flexible intradomain segment between the membranous and the lumenal domains.
TL;DR: Observations indicate that association with negatively charged membranes induces a conformational change within α-lactalbumin to a flexible, molten globule-like state.
TL;DR: Three-dimensional reconstructions from electron micrographs show that a bridge of density exists between the two strands of the filament in Ca(2+)-actin that is absent in Mg(2)+-actin, and may be induced by myosin binding, since this movement generates changes in actin's diffraction that are very similar to the changes in the muscle X-ray pattern during activation.
TL;DR: In this article, the morphologies of small proteins Zn2Cd5-metallothionein, cytochrome c3 and β-lactamase I were studied by high-resolution transmission electron microscopy (HRTEM).
Abstract: The morphologies of the small proteins Zn2Cd5-metallothionein, cytochrome c3 and β-lactamase I immobilized inside carbon nanotubes have been studied by high-resolution transmission electron microscopy (HRTEM); single protein molecules and their associated forms were clearly observed inside the central cavity and a significant amount remained catalytically active indicating that no drastic conformational change had taken place.
TL;DR: It is speculated that the intrinsic loop flexibility of different PTPases may be related to their catalytic rate and may play a role in the wide range of activities observed within this enzyme family.
Abstract: Protein tyrosine phosphatases (PTPases) play critical roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. The structures of several different PTPases have revealed a conserved active site architecture in which a phosphate-binding loop, together with an invariant arginine, cradle the phosphate of a phosphotyrosine substrate and poise it for nucleophilic attack by an invariant cysteine nucleophile. We previously reported that binding of tungstate to the Yop51 PTPase from Yersinia induced a loop conformational change that moved aspartic acid 356 into the active site, where it can function as a general acid. This is consistent with the aspartic acid donating a proton to the tyrosyl leaving group during the initial hydrolysis step. In this report, using a similar structure of the inactive Cys 403-->Ser mutant of the Yersinia PTPase complexed with sulfate, we detail the structural and functional details of this conformational change. In response to oxyanion binding, small perturbations occur in active site residues, especially Arg 409, and trigger the loop to close. Interestingly, the peptide bond following Asp 356 has flipped to ligate a buried, active site water molecule that also hydrogen bonds to the bound sulfate anion and two invariant glutamines. Loop closure also significantly decreases the solvent accessibility of the bound oxyanion and could effectively shield catalytic intermediates from phosphate acceptors other than water. We speculate that the intrinsic loop flexibility of different PTPases may be related to their catalytic rate and may play a role in the wide range of activities observed within this enzyme family.
TL;DR: Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction, and data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5.
TL;DR: The use of limited proteolysis by trypsin and ion spray mass spectrometry is described to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation and it is proposed that this loop contacts the alpha subunit of RNA polymerase to activate transcription.
Abstract: Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.
TL;DR: Investigation of plasmas of patients with the Val Met or Pro Ser substitutions showed that these dysfunctional proteins circulate at low levels and are recognized by the complex-specific monoclonal antibody, which strongly indicates a conformational change as a result of these carboxyl-terminal substitutions.
TL;DR: NMR titration data obtained with both aptamers and their cognate ligands confirm the proposed conformational changes and indicate the formation of a 1:1 complex of RNA:amino acid.
Abstract: In a recent study, an RNA aptamer for the specific recognition of the amino acid L-arginine was evolved from an in vitro selected L-citrulline binding parent sequence [M. Famulok (1994) J. Am. Chem. Soc. 116, 1698-1706]. We have now carried out a structural analysis of these aptamers by using chemical modification experiments. Footprinting experiments and a damage selection approach were performed to identify those positions protected from modification in the presence of the amino acids and modifications that interfere with the binding of the ligand. It is shown that of the two bulged regions present in both aptamers one can be modified without loss of binding activity whereas in the other bulge nearly every position is shown to be involved in the recognition of the ligands. This might be indicative for non-canonical base pairing to occur within the non-Watson-Crick paired regions which might be stabilized by the complexed amino acid. Binding to the cognate amino acid significantly enhances the conformational stability of the RNA. We also tested the sensitivity of both aptamers towards lead (II) ion induced cleavage and identified a hypersensitive cleavage site within the invariant bulged region. Lead cleavage is inhibited by the complexed amino acid, indicating a conformational change of the aptamer upon ligand binding. NMR titration data obtained with both aptamers and their cognate ligands confirm the proposed conformational changes and indicate the formation of a 1:1 complex of RNA:amino acid.
TL;DR: The overall folding of rat cathepsin B was shown to be very similar to that of the human liver enzyme, and the structure of the complex between the enzyme and inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethylketone showed that very little conformational change occurs in the enzyme upon inhibitor binding.
TL;DR: A protein fold, six parallel β strands surrounding the central α helix, is likely to be a common structure in protein families known to have a typical set of nucleotide binding consensus sequenced motifs A and B and to catalyze ATP‐triggered reactions.