Showing papers on "Conformational change published in 2009"
TL;DR: A mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a flux-based approach.
Abstract: The mechanism of ligand binding coupled to conformational changes in macromolecules has recently attracted considerable interest. The 2 limiting cases are the "induced fit" mechanism (binding first) or "conformational selection" (conformational change first). Described here are the criteria by which the sequence of events can be determined quantitatively. The relative importance of the 2 pathways is determined not by comparing rate constants (a common misconception) but instead by comparing the flux through each pathway. The simple rules for calculating flux in multistep mechanisms are described and then applied to 2 examples from the literature, neither of which has previously been analyzed using the concept of flux. The first example is the mechanism of conformational change in the binding of NADPH to dihydrofolate reductase. The second example is the mechanism of flavodoxin folding coupled to binding of its cofactor, flavin mononucleotide. In both cases, the mechanism switches from being dominated by the conformational selection pathway at low ligand concentration to induced fit at high ligand concentration. Over a wide range of conditions, a significant fraction of the flux occurs through both pathways. Such a mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a flux-based approach.
496 citations
TL;DR: A novel biomimetic nanochannel system which has an ion concentration effect that provides a nonlinear response to potassium ion at the concentration ranging from 0 to 1500 microM and can be further generalized to other more complicated functional molecules for the exploitation of novel bioinspired intelligent nanopore machines.
Abstract: Potassium is especially crucial in modulating the activity of muscles and nerves whose cells have specialized ion channels for transporting potassium. Normal body function extremely depends on the regulation of potassium concentrations inside the ion channels within a certain range. For life science, undoubtedly, it is significant and challenging to study and imitate these processes happening in living organisms with a convenient artificial system. Here we report a novel biomimetic nanochannel system which has an ion concentration effect that provides a nonlinear response to potassium ion at the concentration ranging from 0 to 1500 μM. This new phenomenon is caused by the G-quadruplex DNA conformational change with a positive correlation with ion concentration. In this work, G-quadruplex DNA was immobilized onto a synthetic nanopore, which undergoes a potassium-responsive conformational change and then induces the change in the effective pore size. The responsive ability of this system can be regulated by...
319 citations
TL;DR: Simulation of the prion‐derived 8‐residue amyloidogenic peptide and its variant have indicated that an octamer is stable enough to be a seed and that the driving force for stabilization is the hydrophobic effect.
Abstract: The aggregation observed in protein conformational diseases is the outcome of significant new β-sheet structure not present in the native state. Peptide model systems have been useful in studies of fibril aggregate formation. Experimentally, it was found that a short peptide AGAAAAGA is one of the most highly amyloidogenic peptides. This peptide corresponds to the Syrian hamster prion protein (ShPrP) residues 113–120. The peptide was observed to be conserved in all species for which the PrP sequence has been determined. We have simulated the stabilities of oligomeric AGAAAAGA and AAAAAAAA (A8) by molecular dynamic simulations. Oligomers of both AGAAAAGA and AAAAAAAA were found to be stable when the size is 6 to 8 (hexamer to octamer). Subsequent simulation of an additional α-helical AAAAAAAA placed on the A8-octamer surface has revealed molecular events related to conformational change and oligomer growth. Our study addresses both the minimal oligomeric size of an aggregate seed and the mechanism of seed growth. Our simulations of the prion-derived 8-residue amyloidogenic peptide and its variant have indicated that an octamer is stable enough to be a seed and that the driving force for stabilization is the hydrophobic effect.
177 citations
TL;DR: The interconversion between newly identified Ras conformations revealed by this study advances mechanistic understanding of Ras function and provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras.
Abstract: Ras mediates signaling pathways controlling cell proliferation and development by cycling between GTP- and GDP-bound active and inactive conformational states. Understanding the complete reaction path of this conformational change and its intermediary structures is critical to understanding Ras signaling. We characterize nucleotide-dependent conformational transition using multiple-barrier-crossing accelerated molecular dynamics (aMD) simulations. These transitions, achieved for the first time for wild-type Ras, are impossible to observe with classical molecular dynamics (cMD) simulations due to the large energetic barrier between end states. Mapping the reaction path onto a conformer plot describing the distribution of the crystallographic structures enabled identification of highly populated intermediate structures. These structures have unique switch orientations (residues 25–40 and 57–75) intermediate between GTP and GDP states, or distinct loop3 (46–49), loop7 (105–110), and α5 C-terminus (159–166) conformations distal from the nucleotide-binding site. In addition, these barrier-crossing trajectories predict novel nucleotide-dependent correlated motions, including correlations of α2 (residues 66–74) with α3-loop7 (93–110), loop2 (26–37) with loop10 (145–151), and loop3 (46–49) with α5 (152–167). The interconversion between newly identified Ras conformations revealed by this study advances our mechanistic understanding of Ras function. In addition, the pattern of correlated motions provides new evidence for a dynamic linkage between the nucleotide-binding site and the membrane interacting C-terminus critical for the signaling function of Ras. Furthermore, normal mode analysis indicates that the dominant collective motion that occurs during nucleotide-dependent conformational exchange, and captured in aMD (but absent in cMD) simulations, is a low-frequency motion intrinsic to the structure.
172 citations
TL;DR: The binding kinetics in a simple four‐state model of ligand–protein binding is considered, which finds a simple, characteristic difference between the on‐ and off‐rates in the two mechanisms if the conformational relaxation into the ground states is fast.
Abstract: The binding of a ligand molecule to a protein is often accompanied by conformational changes of the protein. A central question is whether the ligand induces the conformational change (induced-fit), or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of ground and excited states of the protein (selected-fit). We consider here the binding kinetics in a simple four-state model of ligand-protein binding. In this model, the protein has two conformations, which can both bind the ligand. The first conformation is the ground state of the protein when the ligand is off, and the second conformation is the ground state when the ligand is bound. The induced-fit mechanism corresponds to ligand binding in the unbound ground state, and the selected-fit mechanism to ligand binding in the excited state. We find a simple, characteristic difference between the on- and off-rates in the two mechanisms if the conformational relaxation into the ground states is fast. In the case of selected-fit binding, the on-rate depends on the conformational equilibrium constant, whereas the off-rate is independent. In the case of induced-fit binding, in contrast, the off-rate depends on the conformational equilibrium, while the on-rate is independent. Whether a protein binds a ligand via selected-fit or induced-fit thus may be revealed by mutations far from the protein's binding pocket, or other "perturbations" that only affect the conformational equilibrium. In the case of selected-fit, such mutations will only change the on-rate, and in the case of induced-fit, only the off-rate.
153 citations
TL;DR: The results suggest that crystal structures of MsbA capture features of the motion that couples ATP energy expenditure to work, providing a framework for the mechanism of substrate transport.
136 citations
TL;DR: The crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1) is determined, and the protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent.
Abstract: Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the “pH sensor” that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.
130 citations
TL;DR: Comparing the X-ray crystal structures of the reduced and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides, a displacement of heme a(3) involving both the porphyrin ring and the hydroxyl farnesyl tail is observed, accompanied by protein movements in nearby regions, suggesting an access path for protons into the active site.
Abstract: A role for conformational change in the coupling mechanism of cytochrome c oxidase is the subject of controversy. Relatively small conformational changes have been reported in comparisons of reduced and oxidized crystal structures of bovine oxidase but none in bacterial oxidases. Comparing the X-ray crystal structures of the reduced (at 2.15 A resolution) and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides, we observe a displacement of heme a3 involving both the porphyrin ring and the hydroxyl farnesyl tail, accompanied by protein movements in nearby regions, including the mid part of helix VIII of subunit I which harbors key residues of the K proton uptake path, K362 and T359. The conformational changes in the reduced form are reversible upon reoxidation. They result in an opening of the top of the K pathway and more ordered waters being resolved in that region, suggesting an access path for protons into the active site. In all high-resolution structures of oxidized R. sphaeroides cyt...
125 citations
TL;DR: “open” and “closed” CSs in PTE and dominant structural transition in the enzyme that links them are identified and analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.
Abstract: To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier—either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies “open” and “closed” CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.
115 citations
TL;DR: It is shown that MHC-restricted antigen recognition by the alphabeta TCR results in a specific conformational change confined to the A-B loop within the alpha chain of the constant domain (Calpha).
109 citations
TL;DR: In this study, cholesterol was found to stimulate SNARE-mediated lipid mixing of proteoliposomes by a factor of 5 at a physiological concentration, Surprisingly, however, the stimulatory effect was more pronounced when cholesterol was on the v-SNARE side than when it is on the t- SNARE side.
Abstract: Neurotransmitter release at the synapse requires membrane fusion. The SNARE complex, composed of the plasma membrane t-SNAREs syntaxin 1A and SNAP-25 and the vesicle v-SNARE synaptobrevin, mediates the fusion of 2 membranes. Synaptic vesicles contain unusually high cholesterol, but the exact role of cholesterol in fusion is not known. In this study, cholesterol was found to stimulate SNARE-mediated lipid mixing of proteoliposomes by a factor of 5 at a physiological concentration. Surprisingly, however, the stimulatory effect was more pronounced when cholesterol was on the v-SNARE side than when it was on the t-SNARE side. Site-directed spin labeling and both continuous wave (CW) and pulsed EPR revealed that cholesterol induces a conformational change of the v-SNARE transmembrane domain (TMD) from an open scissors-like dimer to a parallel dimer. When the TMD was forced to form a parallel dimer by the disulfide bond, the rate was stimulated 2.3-fold even without cholesterol, supporting the relevance of the open-to-closed conformational change to the fusion activity. The open scissors-like conformation may be unfavorable for fusion and cholesterol may relieve this inhibitory factor.
TL;DR: A spontaneous encapsulation of a globular protein into the CNT was observed through molecular dynamics simulation, and the free energy of the system was found to be decreased after the encapsulation, which is the most fundamental reason for this spontaneous process.
TL;DR: It is found that in four of five simulations of the ADP/ATP‐bound dimer, the relative rotation of the helical and catalytic subdomains in the ADp‐bound monomer results in opening of the ADC‐bound active site, probably sufficient or close to sufficient to allow nucleotide exchange.
Abstract: ABC transporters are ubiquitous, ATP-dependent transmembrane pumps. The mechanism by which ATP hydrolysis in the nucleotide-binding domain (NBD) effects conformational changes in the transmembrane domain that lead to allocrite translocation remains largely unknown. A possible aspect of this mechanism was suggested by previous molecular dynamics simulations of the MJ0796 NBD dimer, which revealed a novel, nucleotide-dependent intrasubunit conformational change involving the relative rotation of the helical and catalytic subdomains. Here, we find that in four of five simulations of the ADP/ATP-bound dimer, the relative rotation of the helical and catalytic subdomains in the ADP-bound monomer results in opening of the ADP-bound active site, probably sufficient or close to sufficient to allow nucleotide exchange. We also observe that in all five simulations of the ADP/ATP-bound dimer, the intimate contact of the LSGGQ signature sequence with the ATP γ-phosphate is weakened by the intrasubunit conformational change within the ADP-bound monomer. We discuss how these results support a constant contact model for the function of the NBD dimer in contrast to switch models, in which the NBDs are proposed to fully disassociate during the catalytic cycle. Proteins 2009. © 2008 Wiley-Liss, Inc.
TL;DR: Rapid association rates reveal that ligand binding is not dependent upon a slow conformational change in the protein to permit ligand access, despite the closed conformation observed in the NMR and crystal structures, and a binding-competent transition state characterized by increased structural disorder is suggested.
Abstract: Hypoxia inducible factors (HIFs) are heterodimeric transcription factors responsible for the metazoan hypoxia response and are required for tumor growth, metastasis and resistance to cancer treatment. The C-terminal PAS domain of HIF2α (HIF2α PAS-B) contains a preformed solvent-inaccessible cavity that binds artificial ligands that allosterically perturb the formation of the HIF heterodimer. To better understand how small molecules bind within this domain, we examined the structures, equilibrium and transition state thermodynamics of HIF2α PAS-B with several artificial ligands using ITC, NMR exchange spectroscopy and X-ray crystallography. Rapid association rates reveal that ligand binding is not dependent upon a slow conformational change in the protein to permit ligand access, despite the closed conformation observed in NMR and crystal structures. Compensating enthalpic and entropic contributions to the thermodynamic barrier for ligand binding suggest a binding-competent transition state characterized by increased structural disorder. Finally, molecular dynamics simulations reveal conversion between open and closed conformations of the protein and pathways of ligand entry into the binding pocket.
TL;DR: It is proposed that the initial interaction of an antigen with a TCR may influence the conformation of oligomeric TCR complexes so that TCRs act cooperatively to transmit signals from peptide-MHC.
Abstract: The CD3epsilon subunit of the T cell receptor (TCR) complex undergoes a conformational change upon ligand binding that is thought to be important for the activation of T cells. To study this process, we built a molecular dynamics model of the transmission of the conformational change within the ectodomains of CD3. The model showed that the CD3 dimers underwent a stiffening effect that was funneled to the base of the CD3epsilon subunit. Mutation of two relevant amino acid residues blocked transmission of the conformational change and the differentiation and activation of T cells. Furthermore, this inhibition occurred even in the presence of excess endogenous CD3epsilon subunits. These results emphasize the importance of the conformational change in CD3epsilon for the activation of T cells and suggest the existence of unforeseen cooperativity between TCR complexes.
TL;DR: It is reported that cAMP also induces the translocation of Epac1 toward the plasma membrane, which enhances its ability to induce Rap-mediated cell adhesion and its recruitment to the plasma membranes.
Abstract: Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion and cell-cell junction formation. Here, we report that cAMP also induces the translocation of Epac1 toward the plasma membrane. Combining high-resolution confocal fluorescence microscopy with total internal reflection fluorescence and fluorescent resonance energy transfer assays, we observed that Epac1 translocation is a rapid and reversible process. This dynamic redistribution of Epac1 requires both the cAMP-induced conformational change as well as the DEP domain. In line with its translocation, Epac1 activation induces Rap activation predominantly at the plasma membrane. We further show that the translocation of Epac1 enhances its ability to induce Rap-mediated cell adhesion. Thus, the regulation of Epac1-Rap signaling by cAMP includes both the release of Epac1 from autoinhibition and its recruitment to the plasma membrane.
TL;DR: RovA is established as an intrinsic temperature-sensing protein in which thermally induced conformational changes interfere with DNA-binding capacity, and secondarily render RovA susceptible to proteolytic degradation.
Abstract: Pathogens, which alternate between environmental reservoirs and a mammalian host, frequently use thermal sensing devices to adjust virulence gene expression. Here, we identify the Yersinia virulence regulator RovA as a protein thermometer. Thermal shifts encountered upon host entry lead to a reversible conformational change of the autoactivator, which reduces its DNA-binding functions and renders it more susceptible for proteolysis. Cooperative binding of RovA to its target promoters is significantly reduced at 37°C, indicating that temperature control of rovA transcription is primarily based on the autoregulatory loop. Thermally induced reduction of DNA-binding is accompanied by an enhanced degradation of RovA, primarily by the Lon protease. This process is also subject to growth phase control. Studies with modified/chimeric RovA proteins indicate that amino acid residues in the vicinity of the central DNA-binding domain are important for proteolytic susceptibility. Our results establish RovA as an intrinsic temperature-sensing protein in which thermally induced conformational changes interfere with DNA-binding capacity, and secondarily render RovA susceptible to proteolytic degradation.
TL;DR: It is demonstrated that mutation of a conserved glycine in the linker region inactivates FtsH and visualizes an inward movement of the aromatic pore residues and generates a model of substrate translocation by AAA+ proteases.
Abstract: The hexameric membrane-spanning ATP-dependent metalloprotease FtsH is universally conserved in eubacteria, mitochondria, and chloroplasts, where it fulfills key functions in quality control and signaling. As a member of the self-compartmentalizing ATPases associated with various cellular activities (AAA+ proteases), FtsH converts the chemical energy stored in ATP via conformational rearrangements into a mechanical force that is used for substrate unfolding and translocation into the proteolytic chamber. The crystal structure of the ADP state of Thermotoga maritima FtsH showed a hexameric assembly consisting of a 6-fold symmetric protease disk and a 2-fold symmetric AAA ring. The 2.6 A resolution structure of the cytosolic region of apo-FtsH presented here reveals a new arrangement where the ATPase ring shows perfect 6-fold symmetry with the crucial pore residues lining an open circular entrance. Triggered by this conformational change, a substrate-binding edge beta strand appears within the proteolytic domain. Comparison of the apo- and ADP-bound structure visualizes an inward movement of the aromatic pore residues and generates a model of substrate translocation by AAA+ proteases. Furthermore, we demonstrate that mutation of a conserved glycine in the linker region inactivates FtsH.
TL;DR: The conclusions indicate that the sole mobility of α9 helix side‐chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by α5 helix movement.
Abstract: The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so-called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position-restrained MD simulations. Our conclusions indicate that the sole mobility of alpha9 helix side-chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by alpha5 helix movement. The role of selected alpha5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase.
TL;DR: Comparison of the structures of the liganded and unliganded CAP suggests that cAMP stabilizes the active DNA binding conformation of CAP through the interactions that the N6 of the adenosine makes with the C-helices.
Abstract: The binding of cAMP to the Escherichia coli catabolite gene activator protein (CAP) produces a conformational change that enables it to bind specific DNA sequences and regulate transcription, which it cannot do in the absence of the nucleotide. The crystal structures of the unliganded CAP containing a D138L mutation and the unliganded WT CAP were determined at 2.3 and 3.6 A resolution, respectively, and reveal that the two DNA binding domains have dimerized into one rigid body and their two DNA recognition helices become buried. The WT structure shows multiple orientations of this rigid body relative to the nucleotide binding domain supporting earlier biochemical data suggesting that the inactive form exists in an equilibrium among different conformations. Comparison of the structures of the liganded and unliganded CAP suggests that cAMP stabilizes the active DNA binding conformation of CAP through the interactions that the N 6 of the adenosine makes with the C-helices. These interactions are associated with the reorientation and elongation of the C-helices that precludes the formation of the inactive structure.
TL;DR: A mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS) to propose that Ca2+-binding to CBD2 induces a second electrostatic switch, required to alleviate Na+-dependent inactivation of Na+/Ca2+ exchange.
Abstract: Ion transport performed by the Na+/Ca2+ exchanger (NCX) is regulated via its cytosolic Ca2+-binding domains, CBD1 and CBD2, which act as sensors for intracellular Ca2+. Striking differences in the electrostatic potential of the Ca2+-bound and Ca2+-free forms turn the CBD1 and CBD2 Ca2+-binding sites into electrostatic switches similar to those of C2 domains. Binding of Ca2+ with high affinity to CBD1 induces a conformational change that is relayed to the transmembrane domain and thereby initiates Na+/Ca2+ exchange. The Ca2+ concentration at which this conformational change occurs is determined by the Ca2+ affinities of the strictly conserved CBD1 Ca2+-binding sites that are modulated by an adjacent, variable region of CBD2. In contrast, the Ca2+-binding properties of CBD2 depend on the isoform and the type of residues in the Ca2+-binding sites, encoded by a mutually exclusive exon. This second electrostatic switch, formed by CBD2, appears to be required for sustained Na+/Ca2+ exchange and may allow tailored, tissue-specific exchange activities.
TL;DR: Together, these NMR and biochemical experiments provide additional insight into the mechanism of millisecond motions in the RNase A catalytic cycle.
Abstract: The role of the flexible loop 1 in protein conformational motion and the dissociation of enzymatic product from Ribonuclease A (RNase A) was investigated by creation of a chimeric enzyme in which a six residue loop 1 from the RNase A homolog, eosinophil cationic protein (ECP) replaced the twelve residue loop in RNase A. The chimera (RNase AECP) experiences only local perturbations in NMR backbone chemical shifts compared to WT RNase A. Many of the flexible residues that were previously identified in WT as involved in an important conformational change now experience no NMR-detected millisecond motions in the chimera. Likewise, binding of the product analog, 3′-CMP to RNase AECP results in only minor chemical shift changes in the enzyme similar to what is observed for the H48A mutant of RNase A and in contrast to WT enzyme. For both RNase AECP and H48A there is a 10-fold decrease in the product release rate constant, koff compared to WT and in agreement with previous studies indicating the importance of flexibility in RNase A in the overall rate-limiting product release step. Together these NMR and biochemical experiments provide additional insight into the mechanism of millisecond motions in the RNase A catalytic cycle.
TL;DR: Results collectively suggest that 4-HPR-induced apoptosis is associated with a ROS-mediated conformational change in Akt, and this change mediates dephosphorylation of Akt via regulating Akt-Hsp90 orAkt-PP2A complex formation.
TL;DR: The QCM-D model resulted in decreased adsorbed mass with increased polymer chain flexibility, probably due to polymer chain rearrangement rather than protein conformational change, and fibrinogen maintained a more native conformation on the flexible polymer.
Abstract: By combining quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR), the organic mass, water content, and corresponding protein film structure of fibrinogen adsorbed to acrylic polymeric substrates with varying polymer chain flexibility was investigated. Albumin and immunoglobulin G were included as reference proteins. For fibrinogen, the QCM-D model resulted in decreased adsorbed mass with increased polymer chain flexibility. This stands in contrast to the SPR model, in which the adsorbed mass increased with increased polymer chain flexibility. As the QCM-D model includes the hydrodynamically coupled water, we propose that on the nonflexible polymer significant protein conformational change with water incorporation in the protein film takes place. Fibrinogen maintained a more native conformation on the flexible polymer, probably due to polymer chain rearrangement rather than protein conformational change. In comparison with immunoglobulin G and albumin, polymer chain flexibility had only minor impact on adsorbed mass and protein structure. Understanding the adsorption and corresponding conformational change of a protein together with the mutual rearrangement of the polymer chain upon adsorption not only has implications in biomaterial science but could also increase the efficacy of molecular imprinted polymers (MIPs).
TL;DR: Results suggest that all peptidic mimics of Tat induce the same dynamics in TAR within this protein binding site, however, the new cyclic peptide mimic of Tat represents a new class of ligands with a unique effect on the dynamics and the structure of the apical loop.
Abstract: The HIV-1 TAR RNA represents a well-known paradigm to study the role of dynamics and conformational change in RNA function. This regulatory RNA changes conformation in response to binding of Tat protein and of a variety of peptidic and small molecule ligands, indicating that its conformational flexibility and intrinsic dynamics play important roles in molecular recognition. We have used (13)C NMR relaxation experiments to examine changes in the motional landscape of HIV-1 TAR in the presence of three ligands of different affinity and specificity. The ligands are argininamide, a linear peptide mimic of the Tat basic domain and a cyclic peptide that potently inhibits Tat-dependent activation of transcription. All three molecules induce the same motional characteristics within the three nucleotides bulge that represents the Tat-binding site. However, the cyclic peptide has a unique motional signature in the apical loop, which represents a binding site for the essential host co-factor cyclin T1. These results suggest that all peptidic mimics of Tat induce the same dynamics in TAR within this protein binding site. However, the new cyclic peptide mimic of Tat represents a new class of ligands with a unique effect on the dynamics and the structure of the apical loop.
TL;DR: A molecular-dynamics perturbation method, the Rotamerically Induced Perturbation (RIP), is introduced, which can generate large, coherent motions of structural elements in picoseconds by applying large torsional perturbations to individual sidechains.
Abstract: Protein conformational changes and dynamic behavior are fundamental for such processes as catalysis, regulation, and substrate recognition. Although protein dynamics have been successfully explored in computer simulation, there is an intermediate-scale of motions that has proven difficult to simulate—the motion of individual segments or domains that move independently of the body the protein. Here, we introduce a molecular-dynamics perturbation method, the Rotamerically Induced Perturbation (RIP), which can generate large, coherent motions of structural elements in picoseconds by applying large torsional perturbations to individual sidechains. Despite the large-scale motions, secondary structure elements remain intact without the need for applying backbone positional restraints. Owing to its computational efficiency, RIP can be applied to every residue in a protein, producing a global map of deformability. This map is remarkably sparse, with the dominant sites of deformation generally found on the protein surface. The global map can be used to identify loops and helices that are less tightly bound to the protein and thus are likely sites of dynamic modulation that may have important functional consequences. Additionally, they identify individual residues that have the potential to drive large-scale coherent conformational change. Applying RIP to two well-studied proteins, Dihdydrofolate Reductase and Triosephosphate Isomerase, which possess functionally-relevant mobile loops that fluctuate on the microsecond/millisecond timescale, the RIP deformation map identifies and recapitulates the flexibility of these elements. In contrast, the RIP deformation map of α-lytic protease, a kinetically stable protein, results in a map with no significant deformations. In the N-terminal domain of HSP90, the RIP deformation map clearly identifies the ligand-binding lid as a highly flexible region capable of large conformational changes. In the Estrogen Receptor ligand-binding domain, the RIP deformation map is quite sparse except for one large conformational change involving Helix-12, which is the structural element that allosterically links ligand binding to receptor activation. RIP analysis has the potential to discover sites of functional conformational changes and the linchpin residues critical in determining these conformational states.
TL;DR: Two sequential conformational changes in the protein in response to pH or substrate ions are identified by cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH.
TL;DR: It is reported that binding of Hsp90 to tau facilitates a conformational change that could result in its phosphorylation by glycogen synthase kinase 3 and its aggregation into filamentous structures.
Abstract: Tau pathology, associated with Alzheimer's disease, is characterized by the presence of phosphorylated and aggregated tau. Phosphorylation of tau takes place mainly in the vicinity of the tubulin-binding region of the molecule and its self aggregation is also mediated via this tubulin-binding region. Tau phosphorylation and aggregation have been related with conformational changes of the protein. These changes could be regulated by chaperones such as heat shock proteins, since one of these, heat shock protein 90 (Hsp90), has already been described as a putative tau-binding protein. In this work, we have confirmed the interaction of Hsp90 with tau protein and report that binding of Hsp90 to tau facilitates a conformational change that could result in its phosphorylation by glycogen synthase kinase 3 and its aggregation into filamentous structures.
TL;DR: The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the 1 protein, each complexed with three copies of the protector protein 3.
Abstract: The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the 1 protein, each complexed with three copies of the protector protein 3. Conformational changes in 1 following the proteolytic removal of 3 lead to release of the myristoylated N-terminal cleavage fragment 1N and ultimately to membrane penetration. The 1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant 1 trimers and synthetic 1N peptides with both RBCs and liposomes. The 1 trimer mediates hemolysis and liposome disruption under conditions that promote the 1 conformational change, and mutations that inhibit 1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free 1 trimer. Autolytic cleavage to form 1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that 1N cleavage is not required when steric barriers are removed. Synthetic myristoylated 1N peptide forms size-selective pores in liposomes, as measured by fluorescence dequenching of labeled dextrans of different sizes. Addition of a C-terminal solubility tag to the peptide does not affect activity, but sequence substitution V13N or L36D reduces liposome disruption. These substitutions are in regions of alternating hydrophobic residues. Their locations, the presence of an N-terminal myristoyl group, and the full activity of a C-terminally extended peptide, along with circular dichroism data that indicate prevalence of -strand secondary structure, suggest a model in which 1N -hairpins assemble in the membrane to form a -barrel pore.
TL;DR: Activation, deactivation, and sensitization time courses and amplitudes were used to derive a kinetic scheme and rate constants, from which the EC50 and frequency dependence of mGluR1 activation under non-steady-state conditions, as occurs during synaptic transmission are inferred.
Abstract: Metabotropic glutamate receptor (mGluR) activation has been extensively studied under steady-state conditions However, at central synapses, mGluRs are exposed to brief submillisecond glutamate transients and may not reach steady-state The lack of information on the kinetics of mGluR activation impairs accurate predictions of their operation during synaptic transmission Here, we report experiments designed to investigate mGluR kinetics in real-time We inserted either CFP or YFP into the second intracellular loop of mGluR1β When these constructs were coexpressed in PC12 cells, glutamate application induced a conformational change that could be monitored, using fluorescence resonance energy transfer (FRET), with an EC50 of 75 μM The FRET response was mimicked by the agonist DHPG, abolished by the competitive antagonist MCPG, and partially inhibited by mGluR1-selective allosteric modulators These results suggest that the FRET response reports active conformations of mGluR1 dimers The solution exchange at the cell membrane was optimized for voltage-clamped cells by recording the current induced by co-application of 30 mM potassium When glutamate was applied at increasing concentrations up to 2 mM, the activation time course decreased to a minimum of approximately 10 ms, whereas the deactivation time course remained constant (∼50 ms) During long-lasting applications, no desensitization was observed In contrast, we observed a robust sensitization of the FRET response that developed over approximately 400 ms Activation, deactivation, and sensitization time courses and amplitudes were used to derive a kinetic scheme and rate constants, from which we inferred the EC50 and frequency dependence of mGluR1 activation under non-steady-state conditions, as occurs during synaptic transmission