Showing papers on "Conformational change published in 1992"
TL;DR: The crystal structure of an extracellular triglyceride lipase inhibited irreversibly by diethyl p-nitrophenyl phosphate (E600) was solved by X-ray crystallographic methods and refined to a resolution of 2.65 A.
Abstract: The crystal structure of an extracellular triglyceride lipase (from a fungus Rhizomucor miehei) inhibited irreversibly by diethyl p-nitrophenyl phosphate (E600) was solved by X-ray crystallographic methods and refined to a resolution of 2.65 A. The crystals are isomorphous with those of n-hexylphosphonate ethyl ester/lipase complex [Brzozowski, A. M., Derewenda, U., Derewenda, Z. S., Dodson, G. G., Lawson, D. M., Turkenburg, J. P., Bjorkling, F., Huge-Jensen, B., Patkar, S. A., & Thim, L. (1991) Nature 351, 491-494], where the conformational change was originally observed. The higher resolution of the present study allowed for a detailed analysis of the stereochemistry of the change observed in the inhibited enzyme. The movement of a 15 amino acid long "lid" (residues 82-96) is a hinge-type rigid-body motion which transports some of the atoms of a short alpha-helix (residues 85-91) by over 12 A. There are two hinge regions (residues 83-84 and 91-95) within which pronounced transitions of secondary structure between alpha and beta conformations are caused by dramatic changes of specific conformational dihedral angles (phi and psi). As a result of this change a hydrophobic area of ca. 800 A2 (8% of the total molecule surface) becomes exposed. Other triglyceride lipases are also known to have "lids" similar to the one observed in the R. miehei enzyme, and it is possible that the general stereochemistry of lipase activation at the oil-water interfaces inferred from the present X-ray study is likely to apply to the entire family of lipases.
492 citations
TL;DR: The structure of the unliganded form of the binding protein refined to 1.8-A resolution, combined with that for the liganded form, provides the first crystallographic evidence that a major ligand-induced conformational change occurs in a periplasmic binding protein.
Abstract: The periplasmic maltodextrin binding protein of Escherichia coli serves as an initial receptor for the active transport of and chemotaxis toward maltooligosaccharides. The three-dimensional structure of the binding protein complexed with maltose has been previously reported [Spurlino, J. C., Lu, G.-Y., & Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219]. Here we report the structure of the unliganded form of the binding protein refined to 1.8-A resolution. This structure, combined with that for the liganded form, provides the first crystallographic evidence that a major ligand-induced conformational change occurs in a periplasmic binding protein. The unliganded structure shows a rigid-body "hinge-bending" between the two globular domains by approximately 35 degrees, relative to the maltose-bound structure, opening the sugar binding site groove located between the two domains. In addition, there is an 8 degrees twist of one domain relative to the other domain. The conformational changes observed between this structure and the maltose-bound structure are consistent with current models of maltose/maltodextrin transport and maltose chemotaxis and solidify a mechanism for receptor differentiation between the ligand-free and ligand-bound forms in signal transduction.
480 citations
TL;DR: It is concluded that transcriptional inactivation of steroid receptors by antihormones involves the induction of an inappropriate structural conformation at the extreme carboxyl terminus of the ligand binding domain.
352 citations
TL;DR: The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution.
Abstract: We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances Details of the FRET analysis are presented, features of the analysis with particular relevance to DNA structures are emphasized Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases The mode of ion interaction at the strand exchange site of the junction is discussed
254 citations
TL;DR: Two series of site-directed mutations to the individual Ca(2+)-binding sites of Drosophila melanogaster calmodulin have been generated and studied and it appears that binding of Ca2+ to either carboxyl-terminal site can elicit the first phase of the response but the second phase is almost abolished when site 4 is the mutated site.
216 citations
TL;DR: It is demonstrated that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity.
Abstract: PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.
188 citations
TL;DR: The partial unfolding and concomitant deep insertion of SecA into membranes may aid in the insertion of precursor proteins into the inner membrane and may influence possible interactions between SecA and the integral membrane export machinery components.
178 citations
TL;DR: It is shown that truncation mutants of alpha HL missing 2 to 22 N-terminal amino acids form oligomers on the surfaces of rRBCs but fail to lyse the cells, while mutants missing 3 or 5 amino acids at the C terminus are very inefficient at oligomerization but do lyse rR BCs, albeit extremely slowly.
160 citations
TL;DR: It is concluded that suramin binds to many growth factors and that this induces microaggregation and, in some cases, conformational changes in the growth factor.
Abstract: Suramin inhibits the binding of a variety of growth factors to their cell surface receptors. The direct interaction of suramin with acidic fibroblast growth factor has been detected by the enhancement of the drug's fluorescence in the presence of the protein with the maximum effect occurring at a molar ratio of suramin to aFGF of 2:1. This interaction stabilizes aFGF to thermal denaturation and partially protects a free thiol in its polyanion binding site from oxidation. The binding of suramin to aFGF also induces aggregation of the growth factor to at least a hexameric state as detected by static and dynamic light scattering as well as by gel filtration studies. Both CD and amide I' FTIR spectra of aFGF in the presence and absence of suramin suggest that the drug may also be causing a small conformational change in the growth factor. Suramin produces an even greater aggregation of bFGF and PDGF but not of EGF or IGF-1. Evidence for a suramin-induced conformational change in IGF-1 but not EGF is found by CD, however. It is concluded that suramin binds to many growth factors and that this induces microaggregation and, in some cases, conformational changes. In the case of aFGF, suramin interacts at or near its heparin binding site. The relationship between these phenomena and the anti-growth factor activity of suramin remains to be clearly elucidated.
147 citations
TL;DR: It is suggested that the conformational change of the protein increases the specificity of receptor binding and that the closed configuration of the iron-loaded protein is one, or perhaps the, decisive step in the mechanism for receptor-mediated endocytosis.
135 citations
TL;DR: Results indicate that DS500 (500-kDa dextran sulfate) induces aggregation of factor XII, and implies that aggregation provides a special mechanism whereby proteolytic cleavage is accomplished efficiently when factor XII molecules are bound side by side on the DS500 molecule.
TL;DR: The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins.
Abstract: Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed.
TL;DR: In stoichiometric amounts, the spin label N‐tempoyl‐(p‐chloromercuribenzamide) reacts rapidly with one cysteine residue in membrane‐bound bovine rhodopsin.
Abstract: — In stoichiometric amounts, the spin label N-tempoyl-(p-chloromercuribenzamide) reacts rapidly with one cysteine residue in membrane-bound bovine rhodopsin. This residue is distinct from the two reactive cysteines previously used as attachment sites for spectroscopic labels, and is on the external surface of the protein near the cytoplasmic membrane/aqueous interface.
The spin-labeled side chain has revealed a light-induced conformational change in membrane-bound rhodopsin that is apparently not associated with protein aggregation. The changes are reversible upon the addition of 11-cis retinal, and the magnitude of the change is dependent on the identity of the phospholipid in the surrounding bilayer. Alteration of lipid composition has a much larger effect on bleached rhodopsin than rhodopsin itself, indicating that the former is more readily deformable in response to changes in bilayer properties. This is consistent with the loss of 11-cis retinal binding energy in opsin compared to rhodopsin. These results provide direct structural evidence that the conformation of a membrane protein can be modulated by the lipid properties.
TL;DR: The results further support a reaction model for serpins in which partial insertion of this loop into the A sheet is required for trapping of a proteinase in a stable complex, and complete insertion is responsible for the conformational change accompanying cleavage of the reactive bond of the inhibitor.
TL;DR: The data imply that steroids induce a conformational change in their receptors which is necessary for events subsequent to DNA binding, most likely for transactivation.
Abstract: Hormones and antihormones induce related, but distinct, conformational changes in the progesterone receptor [Allan, G. F., Leng, X., Tsai, S. Y., Weigel, N. L., Edwards, D. P., Tsai, M.-J. & O'Malley, B. W. (1992) J. Biol. Chem. 267, 19513-19520]. In both cases the conformational change precedes the dissociation of heat shock proteins and binding to DNA. We have now investigated the steps in hormone action which are dependent upon this conformational change. We show that in the absence of ligand, monoclonal antibodies directed against different regions of the progesterone receptor can induce high-affinity binding to its response element in vitro. This antibody-induced DNA binding is presumably facilitated by enhanced dimerization of receptor monomers. However, antibodies do not induce the hormone-specific conformational change in the progesterone receptor and do not induce in vitro transcription by the receptor. In contrast, the antiprogestin ZK98299, which inhibits receptor binding to DNA, fully induces the antihormone-specific conformational change. Thus, our data imply that steroids induce a conformational change in their receptors which is necessary for events subsequent to DNA binding, most likely for transactivation.
TL;DR: During the kinetic mechanism of the reduction of aldehydes by aldose reductase, a slow (kinetically detectable) conformational change in the enzyme occurs upon coenzyme binding, since NADPH and NADP+ bind to the enzyme very tightly.
TL;DR: The binding of heparin to basic fibroblast growth factor induces a small but highly reproducible conformational change observable in the amide I region of the protein's infrared spectrum, and this spectroscopically observable change has allowed us to probe the functional determinants necessary for heParin to bind the bFGF and to induce the observed conformationalchange.
TL;DR: Gel shift assays and cross-linking experiments demonstrated that T3 greatly induced the monomeric binding of the hTR beta produced in Escherichia coli to DNA, suggesting they undergo a ligand-induced conformational change.
Abstract: T3 binds to intranuclear thyroid hormone receptors (TRs) on target DNA elements and exerts profound influences on gene expression by mechanisms not yet characterized. We used gel shift assays and cross-linking experiments to demonstrate that T3 greatly induced the monomeric binding of the hTR beta produced in Escherichia coli to DNA. T3 also increased the gel mobility of these monomer-DNA complexes suggesting they undergo a ligand-induced conformational change. This effect did not depend on the orientation and spacing of the half-site motifs within the DNA structure. In contrast, T3 had diverse effects on the dimeric interaction. T3 increased the dimeric interaction to the palindrome GGTCA.TGACC (an effect lost by spacing the half-sites with 3 base pairs) and decreased the dimeric interaction to the inverted palindrome containing the TGACC.GGTCA motif. Scatchard analyses indicated that the T3 enhancement on binding was due to an increase in the number of TR with high affinity DNA-binding activity and not ...
TL;DR: The characterized Nif- mutant of Azotobacter vinelandii, designated UW91, demonstrates that the substitution of Fe protein residue alanine 157 by serine does not change the Fe protein's ability to function in FeMo cofactor biosynthesis or insertion, and demonstrates that these events do not require the MgATP-induced conformational change, MgatP hydrolysis, or productive electron transfer to the MoFe protein.
TL;DR: The rate of conformational state fluctuations (interconversion between states) was found to be slow on the nanosecond time scale, as expected for a protein with a relatively large interdomain contact area.
Abstract: 3-Phosphoglycerate kinase is composed of two globular domains separated by a wide cleft. The substrate binding sites are situated on the inner surfaces of the two domains. By analogy to other kinases, it has been postulated that the catalytic mechanism of phosphoglycerate kinase involves a hinge bending domain motion that brings the substrates together to allow phosphoryl transfer. To characterize this large-scale conformational change, as well as the dynamics of the unliganded enzyme in solution, we have applied site-directed mutagenesis and time-resolved nonradiative energy transfer techniques. Two genetically engineered cysteines (Cys-135 and Cys-290), one in each of the two domains, were covalently labeled with a donor and acceptor pair of fluorescent probes. Analysis of subnanosecond fluorescence decay curves yielded the equilibrium distribution of interdomain distances. In the absence of substrates, the distribution of distances between the two labeled sites was very broad, with a full width at half maximum estimated as 20 A or broader, indicative of a large number of conformational substates in solution. The mean distance, 31.5 +/- 1 A, was 8 A smaller than in the crystal structure. Upon addition of ATP alone or of ATP and 3-phosphoglycerate, the average distance increased to 38 +/- 1 A and the width of the distribution decreased. Addition of 3-phosphoglycerate alone induced a similar but smaller change. The rate of conformational state fluctuations (interconversion between states) was found to be slow on the nanosecond time scale, as expected for a protein with a relatively large interdomain contact area.
TL;DR: Altered Fe-protein with Glu replacing Asp has been prepared and retains the ability for the initial nucleotide-dependent conformational change, but either MgADP or MgATP can induce the shift and Mg binding to the nucleotide is no longer essential.
Abstract: Electron transfer in nitrogenase involves a gating process initiated by MgATP (magnesium adenosine triphosphate) binding to Fe-protein. The redox site, an 4Fe:4S cluster, is structurally separated from the MgATP binding site. For MgATP hydrolysis to be coupled to electron transfer, a signal transduction mechanism is proposed that is similar to that in guanosine triphosphatase proteins. Based on the three-dimensional structure of Fe-protein, Asp125 is likely to be part of a putative transduction path. Altered Fe-protein with Glu replacing Asp has been prepared and retains the ability for the initial nucleotide-dependent conformational change. However, either MgADP or MgATP can induce the shift and Mg binding to the nucleotide is no longer essential.
TL;DR: It is suggested that exposure of the heparin-binding site and a putative hydrophobic binding site of VN are linked events dependent upon the same conformational change induced by different stimuli, such as coagulation, heating, adsorption to surfaces, or exposure to acids, urea, or other denaturating agents.
TL;DR: The results support the hypothesis that the preferred pathway of indole from solution into the beta-site is via the alpha-site and the interconnecting tunnel and suggest that the altered kinetic behavior observed for thealpha-mutants in the presence of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open to the closed form.
TL;DR: The neu proto‐oncogene may be converted into a dominantly transforming oncogene by a single point mutation and the receptor structures determined experimentally do allow alternative models involving receptor transmembrane region packing.
Abstract: The neu proto-oncogene may be converted into a dominantly transforming oncogene by a single point mutation. Substitution of a valine residue at position 664 in the transmembrane region with glutamic acid activates the tyrosine kinase of the molecule and is associated with increased receptor dimerization. Previously we have proposed a model in which the glutamic acid side chain stabilizes receptor dimerization by hydrogen bonding. Other models have been proposed in which the mutation leads to a conformational change in the transmembrane region mimicking that assumed to occur following binding of a natural ligand. Synthetic peptides representing part of the transmembrane region were prepared. Some residues were replaced with serine in order to improve peptide solubility to allow purification and analysis. Both the peptides containing valine and glutamic acid dissolved in water and in an artificial lipid monolayer. The structures of the peptides were determined by NMR spectroscopy to be alpha-helical. No significant difference in conformation was observed between the two peptides. This result does not support the model proposing a conformational change. The receptor structures determined experimentally do allow alternative models involving receptor transmembrane region packing.
TL;DR: It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.
Abstract: Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 06 microM was determined Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidineHCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 15 M) The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, ie secreted protein acidic and rich in cysteine, and osteonectin) These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis
TL;DR: Spectroscopic changes evidence a heparin--induced conformational change of the inhibitor which buries Trp30 in a very hydrophobic environment and may have important physiologic and therapeutic bearing.
Abstract: Low molecular mass heparin (5.1 kDa) forms a tight complex with mucus proteinase inhibitor, the physiologic neutrophil elastase inhibitor of the upper respiratory tract. This binding strongly enhances the intrinsic fluorescence of the inhibitor and the rate of neutrophil elastase inhibitor association. One mole of this heparin fragment binds 1 mol of inhibitor with a Kd of 50 nM. From the variation of Kd with ionic strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy i due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy is due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. and (iii), about one-third of low quantum yield of Trp30, the single tryptophan residue of the inhibitor, blue-shifts its maximum emission wavelength by 6 nm, decreases the acrylamide quenching rate constant by a factor of 4, and increases the mean intensity weighted lifetime by a factor of 2.5. These important spectroscopic changes evidence a heparin--induced conformational change of the inhibitor which buries Trp30 in a very hydrophobic environment. Heparin accelerates the inhibition of elastase in a concentration-dependent manner. When both enzyme and inhibitor are saturated by the polymer, the second-order association rate constant is 7.7 x 10(7) M-1 s-1, a value that is 27-fold higher than that measured with the free partners. This finding may have important physiologic and therapeutic bearing.
TL;DR: Compared with solution NMR data, structures obtained from single crystal diffraction studies, and with previous reports of the 13C-CP/MAS NMR spectra, the results suggest that, for resonances of carbon atoms close to a centre of significant conformational change, chemical shifts may be predicted on the basis of conformation alone, but for more distant atoms, changes in chemical shift may be masked by the effects of alterations in the local environment.
TL;DR: A mechanism of metal binding to the phosphofragments is proposed which may be relevant to the formation of neurofibrillary tangles in Alzheimer's disease.
TL;DR: The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples and its separation from the closely related N-methionyl variant (Met-hGH).
Abstract: This paper examines the retention behavior of recombinant DNA-derived human growth hormone (rhGH) in reversed-phase chromatography and its separation from the closely related N-methionyl variant (Met-hGH). It is first shown that retention for rhGH decreases with increasing column temperature when 1-propanol (1-PrOH) is used as organic modifier. On the other hand, retention increases with temperature when acetonitrile (CH3CN) is employed. The differences in behavior for the two organic modifiers could be related to conformational changes in the protein as determined by solution and adsorption intrinsic fluorescence spectroscopy. Specifically, desorption and elution of rhGH using 1-PrOH could be correlated with a solvent-induced conformational change, with retention decreasing with increasing temperature due to the increasing ease of structural alteration. On the other hand for CH3CN the increase in retention correlated with temperature rise was related to a partial structural change yielding a more hydrophobic species. In this case, a surface-driven process is suggested. The work then turned to the separation of rhGH and Met-hGH where it was found for both organic modifiers optimum separation occurred at 45 degrees C and pH 6.5. Separate studies revealed that during the conformational change Met-hGH appeared more hydrophobic than rhGH since protein-protein aggregation was observed at a lower 1-PrOH concentration. It is suggested that this hydrophobic difference, which was optimized under the conditions cited above, resulted in the separation. The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples.
TL;DR: It is shown that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation, and that arginine and guanidine induce similar CD changes.
Abstract: Short basic peptides from the HIV Tat protein bind specifically to a bulge region in TAR RNA, with a single arginine residue providing the only sequence-specific contact. The free amino acid arginine also binds specifically to TAR. Previous circular dichroism (CD) experiments suggested that peptide binding induces a conformational change in TAR. Here we confirm this observation using single arginine-containing peptides and show that arginine or guanidine binding also induces a conformational change in TAR. A peptide containing a single arginine within a stretch of histidines (CYHHHRHHHHHA) shows pH-dependent binding and a corresponding change in TAR conformation, as detected by a decrease in the CD signal at 265 nm. Arginine and guanidine, which bind to TAR with apparent Kd's of approximately 1.5 mM, induce similar CD changes. In contrast, lysine, which does not bind specifically to TAR, has no effect. Mutants of TAR that abolish specific binding (a U-->C substitution in the three-nucleotide bulge, a deletion of the bulge, or an A-U to U-A base pair change above the bulge) show no change in the CD signal upon binding of peptides, arginine, or guanidine. The results suggest that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation.