Showing papers on "Conformational change published in 2007"
TL;DR: The structure of a complex between the PKA catalytic subunit and a mutant RI regulatory subunit, RIalpha(91-379:R333K), containing both cAMP-binding domains is reported, providing a molecular mechanism for the ordered and cooperative activation of PKA by cAMP.
313 citations
TL;DR: A coarse grained model is employed to explore the interplay between protein structure, folding and function which is applicable to allosteric or non-allosteric proteins and it is demonstrated that local unfolding may be due, in part, to competing intra-protein interactions.
282 citations
TL;DR: The permissive geometry model provides a molecular mechanism that rationalizes how the information of ligand binding to TCRalphabeta is transmitted to the CD3 subunits and to the intracellular signaling machinery.
244 citations
TL;DR: A general "confine-and-release" framework for free energy calculations that accounts for free energies of conformational change is introduced and illustrated by demonstrating that an umbrella sampling protocol can obtain converged binding free energies that are independent of the starting protein structure and include these conformational changes free energies.
Abstract: Free energy calculations are increasingly being used to estimate absolute and relative binding free energies of ligands to proteins. However, computed free energies often appear to depend on the initial protein conformation, indicating incomplete sampling. This is especially true when proteins can change conformation on ligand binding, as free energies associated with these conformational changes are either ignored or assumed to be included by virtue of the sampling performed in the calculation. Here, we show that, in a model protein system (a designed binding site in T4 lysozyme), conformational changes can make a difference of several kcal/mol in computed binding free energies and that they are neglected in computed binding free energies if the system remains kinetically trapped in a particular metastable state on simulation timescales. We introduce a general “confine-and-release” framework for free energy calculations that accounts for these free energies of conformational change. We illustrate its use...
162 citations
TL;DR: Fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function.
Abstract: We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol ternary complex upon binding of a dNTP substrate. This fluorescence change is believed to reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function. The rate of the conformational change induced by a complementary dNTP substrate was determined by both conventional stopped-flow and high-time-resolution continuous-flow fluorescence measurements at the ensemble-averaged level. The rate of this conformational change is much faster than that of DNA synthesis but is significantly reduced for noncomplementary dNTPs, as revealed by single-molecule measurements. The high level of selectivity of incoming dNTPs pertinent to this conformational change is a major contributor to replicative fidelity.
160 citations
TL;DR: FRET stoichiometry reveals conformational changes of a protein complex in live cells and indicates how motor protein activity is controlled in cells is likely coupled to changes in protein conformation and cargo association.
Abstract: Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)–labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.
151 citations
TL;DR: Structural comparison combined with mutational analysis identifies a likely single‐stranded RNA (ssRNA)‐binding channel, and a cycle of conformational change coupled to ATP binding and hydrolysis that can explain how ATP binding destabilizes ssRNA binding to Upf1p.
Abstract: Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway that recognizes and degrades aberrant mRNAs containing premature stop codons. A critical protein in NMD is Upf1p, which belongs to the helicase super family 1 (SF1), and is thought to utilize the energy of ATP hydrolysis to promote transitions in the structure of RNA or RNA–protein complexes. The crystal structure of the catalytic core of human Upf1p determined in three states (phosphate-, AMPPNP- and ADP-bound forms) reveals an overall structure containing two RecA-like domains with two additional domains protruding from the N-terminal RecA-like domain. Structural comparison combined with mutational analysis identifies a likely single-stranded RNA (ssRNA)-binding channel, and a cycle of conformational change coupled to ATP binding and hydrolysis. These conformational changes alter the likely ssRNA-binding channel in a manner that can explain how ATP binding destabilizes ssRNA binding to Upf1p.
150 citations
TL;DR: The structure of the complex between CA N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N'-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) is determined using a combination of NMR spectroscopy and X-ray crystallography.
144 citations
TL;DR: Results indicate that while the elastic-network model captures the low-frequency vibrational motions of a protein, the roughness in the free energy function introduced by the DWNM can be used to characterize the transition mechanism between protein conformations.
140 citations
TL;DR: This work describes a protein-based dynamic material, the functional nature of which is derived from the conformational properties of the protein calmodulin (CaM), and prepares an engineered version of CaM in which cysteine residues are included in place of tyrosine residues at the ends of the dumbbell-shaped protein and the binding of the CaM units and ligands resulted in an increased cross-linking density and a decreased swelling of the network.
Abstract: The development of materials that undergo shape changes occupies a central theme in materials science and has proven important in several applications. Several classes of hydrogels, which are cross-linked water-soluble polymers, can change their properties (such as, volume, cross-link density) in response to temperature, pH value, or ionic strength. These dynamic hydrogels can also be modified with biochemical moieties to give materials that change their properties in response to proteins and ligands. 6] For example, hydrogels that undergo volume changes because of antigen–antibody and lectin–carbohydrate interactions have been used as biosensors. The underlying principle of operation of dynamic hydrogel materials relates to a change in their physical or chemical cross-linking density in response to environmental cues. An unexplored alternative to these approaches relies on the use of a natural protein that undergoes a conformational change as a mechanism to alter the characteristics of a material. The functional importance of protein motions in biological systems, together with the wide range of protein motions that can be harnessed, offers a flexible approach to the preparation of dynamic materials. We describe herein an example of a protein-based dynamic material, the functional nature of which is derived from the conformational properties of the protein calmodulin (CaM). Calmodulin is a 16.5-kDa protein with two distinct conformational states (Figure 1). In the presence of calcium ions, CaM has an extended, dumbbell-shaped conformation (herein termed “extended CaM”). This calciumbound CaM undergoes a transition from an extended dumbbell to a collapsed conformation (herein termed “collapsed CaM”) upon the binding of ligands, which include certain antipsychotic drugs (such as, trifluoperazine (TFP)), 13,16] peptides, and a variety of proteins . Recently, Daunert and co-workers described a class of hydrogels that incorporate CaM and a small-molecule ligand as pendant moieties within the network. The binding of the CaM units and ligands resulted in an increased cross-linking density and a decreased swelling of the network. This approach is analogous to the development of dynamic hydrogels based on antigen–antibody and lectin–carbohydrate interactions, but differs from our approach in that the dynamic response of the gel is not due primarily to the conformational properties of CaM. We prepared an engineered version of CaM in which cysteine residues are included in place of tyrosine residues at the ends of the dumbbell-shaped protein (CaM Y34C, Y110C). The distance that separates the two cysteine residues is approximately 50 : in the extended conformation, but is decreased to approximately 15 : in the collapsed conformation. We incorporated the CaM building blocks into a poly(ethylene glycol)-based hydrogel by using a four-armed poly(ethylene glycol) (PEG) molecule terminated at each end with an acrylate group. The acrylate groups react selectively with the sulfhydryl groups on the engineered CaM and therefore serve to cross-link the CaM proteins into watersoluble conjugates. The Ellman test, which measures the amount of free sulfhydryl groups, was performed after a period of 5 minutes and showed that the reaction of the PEG tetraacrylate with the engineered CaM was complete (Figure 2a). Furthermore, MALDI-TOF mass spectrometry confirmed the conversion of the free CaM protein into crosslinked products (Figure 2b). The MALDI spectra, for example, showed a diffuse peak at 27 kDa, which represents a conjugate of a single PEG tetraacrylate chain ( 10.2 kDa) and a single CaM molecule (16.5 kDa). This peak is not observed when the PEG acrylate is present in a high concentration, and these data, along with the loss of the sulfhydryl groups, demonstrate the formation of higher-order conjugates by partial cross-linking. To complete the formation of the hydrogel, solutions containing conjugates of the CaM Figure 1. The two conformational states of calmodulin: an extended, dumbbell-shaped conformation in the presence of calcium ions (left), and a collapsed conformation upon binding to a ligand (right). Dark red: hinge region; blue: ends of dumbbell-shaped protein; light-red spheres: acrylate–thiol linkage; black ribbons, : poly(ethylene glycol) moiety.
136 citations
TL;DR: In this paper, a kinetic and structural analysis of ovine PrP oligomerization was performed and three distinct oligomeric species were formed in parallel, independent kinetic pathways, and only the largest oligomer gave rise to fibrillar structures at high concentration.
Abstract: The prion protein (PrP) propensity to adopt different structures is a clue to its biological role. PrP oligomers have been previously reported to bear prion infectivity or toxicity and were also found along the pathway of in vitro amyloid formation. In the present report, kinetic and structural analysis of ovine PrP (OvPrP) oligomerization showed that three distinct oligomeric species were formed in parallel, independent kinetic pathways. Only the largest oligomer gave rise to fibrillar structures at high concentration. The refolding of OvPrP into these different oligomers was investigated by analysis of hydrogen/deuterium exchange and introduction of disulfide bonds. These experiments revealed that, before oligomerization, separation of contacts in the globular part (residues 127-234) occurred between the S1-H1-S2 domain (residues 132-167) and the H2-H3 bundle (residues 174-230), implying a conformational change of the S2-H2 loop (residues 168-173). The type of oligomer to be formed depended on the site where the expansion of the OvPrP monomer was initiated. Our data bring a detailed insight into the earlier conformational changes during PrP oligomerization and account for the diversity of oligomeric entities. The kinetic and structural mechanisms proposed here might constitute a physicochemical basis of prion strain genesis.
TL;DR: NMR studies of full-length KcsA, a prototypical K+ channel, in its open, closed and intermediate states reveal that at least two conformational states exist both in the selectivity filter and near the C-terminal ends of the TM2 helices.
Abstract: K+ channels conduct and regulate K+ flux across the cell membrane. Several crystal structures and biophysical studies of tetrameric ion channels have revealed many of the structural details of ion selectivity and gating. A narrow pore lined with four arrays of carbonyl groups is responsible for ion selectivity, whereas a conformational change of the four inner transmembrane helices (TM2) is involved in gating. We used NMR to examine full-length KcsA, a prototypical K+ channel, in its open, closed and intermediate states. These studies reveal that at least two conformational states exist both in the selectivity filter and near the C-terminal ends of the TM2 helices. In the ion-conducting open state, we observed rapid structural exchange between two conformations of the filter, presumably of low and high K+ affinity, respectively. Such measurements of millisecond-timescale dynamics reveal the basis for simultaneous ion selection and gating.
TL;DR: This study determines conformational changes in beta-arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V(2) vasopressin receptor or the corresponding unphosphorylated peptide (V( 2)Rnp).
TL;DR: The findings reported here are expected to assist in providing a framework for predicting protein-ligand complexes and for template-based prediction of protein function.
TL;DR: The consequences of FtsZ polymer bundling by YgfE and divalent cations are very similar in terms of GTPase activity, bundle morphology and GTP orientation and therefore it is proposed that this conformational change in bound GTP reveals a general mechanism of FTSZ bundling.
TL;DR: Results support a stepwise model for mutant and wild-type p53 nuclear export, in which the conformational change induced by either the cancer-derived mutation or MDM2 binding precedes p53 ubiquitination.
TL;DR: It is shown here that the periplasmic protein CusB from the Cus copper/silver efflux system has a critical role in Cu(I) and Ag(I), and upon binding metal, CUSB undergoes a conformational change to a more compact structure.
TL;DR: It is shown that ubiquitination can also regulate proteins by transiently inactivating enzymatic function through conformational change in a dimeric enzyme, which can be reversed upon deubiquitination.
Abstract: Ubiquitination is a critical posttranslational regulator of protein stability and/or subcellular localization. Here we show that ubiquitination can also regulate proteins by transiently inactivating enzymatic function through conformational change in a dimeric enzyme, which can be reversed upon deubiquitination. Our model system is the thyroid hormone-activating type 2 deiodinase (D2), an endoplasmic reticulum-resident type 1 integral membrane enzyme. D2 exists as a homodimer maintained by interacting surfaces at its transmembrane and globular cytosolic domains. The D2 dimer associates with the Hedgehog-inducible ubiquitin ligase WSB-1, the ubiquitin conjugase UBC-7, and VDU-1, a D2-specific deubiquitinase. Upon binding of T4, its natural substrate, D2 is ubiquitinated, which inactivates the enzyme by interfering with D2's globular interacting surfaces that are critical for dimerization and catalytic activity. This state of transient inactivity and change in dimer conformation persists until deubiquitination. The continuous association of D2 with this regulatory protein complex supports rapid cycles of deiodination, conjugation to ubiquitin, and enzyme reactivation by deubiquitination, allowing tight control of thyroid hormone action.
TL;DR: The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state.
TL;DR: PYP is an ideal model for protein conformational changes that result in functional change, responding to stimuli and expressing physiological activity, and is reviewed in this paper.
Abstract: Photoactive yellow protein (PYP) is a water-soluble photosensor protein found in purple photosynthetic bacteria. Unlike bacterial rhodopsins, photosensor proteins composed of seven transmembrane helices and a retinal chromophore in halophilic archaebacteria, PYP is a highly soluble globular protein. The α/β fold structure of PYP is a structural prototype of the PAS domain superfamily, many members of which function as sensors for various kinds of stimuli. To absorb a photon in the visible region, PYP has a p-coumaric acid chromophore binding to the cysteine residue via a thioester bond. It exists in a deprotonated trans form in the dark. The primary photochemical event is photo-isomerization of the chromophore from trans to cis form. The twisted cis chromophore in early intermediates is relaxed and finally protonated. Consequently, the chromophore becomes electrostatically neutral and rearrangement of the hydrogen-bonding network triggers overall structural change of the protein moiety, in which local conformational change around the chromophore is propagated to the N-terminal region. Thus, it is an ideal model for protein conformational changes that result in functional change, responding to stimuli and expressing physiological activity. In this paper, recent progress in investigation of the photoresponse of PYP is reviewed.
TL;DR: The crystal structures of the reduced and oxidized form of Populus trichocarpaxdeltoides GPX5 (PtGPX5) are determined, using a selenomethionine derivative, and it is shown that a drastic conformational change is necessary to bring the two distant cysteine residues together to form an intramolecular disulfide bond.
TL;DR: Results confirm that the helical N terminus of AS can mediate specific interactions with other proteins and suggest that membrane binding may regulate the physiological activity of AS in vivo.
TL;DR: Current evidence suggests that TCR triggering is initiated by a combination of segregation and conformational change, with subsequent aggregation contributing to amplification of the signal.
TL;DR: The structure of FXYD1 provides the foundation for understanding its intra-membrane association with the Na,K-ATPase alpha subunit and suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein to modulate its interaction with thealpha subunit.
Abstract: FXYD1 is a major regulatory subunit of the Na,K-ATPase, and the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinases A and C in heart and skeletal muscle sarcolemma. It is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here we present the three-dimensional structure of FXYD1 determined in micelles by NMR spectroscopy. Structure determination was made possible by measuring residual dipolar couplings in weakly oriented micelle samples of the protein. This allowed us to obtain the relative orientations of the helical segments of the protein, and also provided information about the protein dynamics. The structural analysis was further facilitated by the inclusion of distance restraints, obtained from paramagnetic spin label relaxation enhancements, and by refinement with a micelle depth restraint, derived from paramagnetic Mn line broadening effects. The structure of FXYD1 provides the foundation for understanding its intra-membrane association with the Na,K-ATPase α subunit, and suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein, to modulate its interaction with the α subunit.
TL;DR: SAS-based solution structures of the ATPase domain of the EBP NtrC1 from Aquifex aeolicus in different nucleotide states indicate that ATP binding promotes a conformational change that stabilizes complexes between EBPs and sigma54, while subsequent hydrolysis and phosphate release drive the conformationalchange needed to open the polymerase/promoter complex.
TL;DR: Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actsin polymerization.
Abstract: A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.
TL;DR: The recent development of analytic high-throughput technologies enables us to take a bird's view of how metabolism is regulated in real time, with major advantages of FRET nanosensors, which yield data with cellular and subcellular resolution and the method is minimally invasive.
TL;DR: The B→A transition is found to be a quasi-harmonic process, which follows closely the first spontaneous deformation mode of B-DNA, showing that a physiologically-relevant deformation is in coded in the flexibility pattern of DNA.
Abstract: We explore here the possibility of determining theoretically the free energy change associated with large conformational transitions in DNA, like the solvent-induced B⇔A conformational change. We find that a combination of targeted molecular dynamics (tMD) and the weighted histogram analysis method (WHAM) can be used to trace this transition in both water and ethanol/water mixture. The pathway of the transition in the A→B direction mirrors the B→A pathway, and is dominated by two processes that occur somewhat independently: local changes in sugar puckering and global rearrangements (particularly twist and roll) in the structure. The B→A transition is found to be a quasi-harmonic process, which follows closely the first spontaneous deformation mode of B-DNA, showing that a physiologically-relevant deformation is in coded in the flexibility pattern of DNA.
TL;DR: In this paper, site-directed mutagenized cysteine-->serine variants were generated and analyzed for enzymatic and conformational properties under reducing and oxidizing conditions.
Abstract: Cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved cysteine residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast-located CYP20-3, site-directed mutagenized cysteine-->serine variants were generated and analysed for enzymatic and conformational properties under reducing and oxidizing conditions. Compared with the wild-type form, elimination of three out of the four cysteine residues decreased the catalytic efficiency of PPI (peptidyl-prolyl cis-trans isomerase) activity of the reduced CYP20-3, indicating a regulatory role of dithiol-disulfide transitions in protein function. Oxidation was accompanied by conformational changes with a predominant role in the structural rearrangement of the disulfide bridge formed between Cys(54) and Cys(171). The rather negative E(m) (midpoint redox potential) of -319 mV places CYP20-3 into the redox hierarchy of the chloroplast, suggesting the activation of CYP20-3 in the light under conditions of limited acceptor availability for photosynthesis as realized under environmental stress. Chloroplast Prx (peroxiredoxins) were identified as interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in the reduction of 2-Cys PrxA and 2-Cys PrxB was assigned to Cys(129) and Cys(171). In addition, it was shown that the isomerization and disulfide-reduction activities are two independent functions of CYP20-3 that both are regulated by the redox state of its active centre.