Showing papers on "Conformational change published in 2005"

Fluorescent Amplifying Recognition for DNA G-Quadruplex Folding with a Cationic Conjugated Polymer: A Platform for Homogeneous Potassium Detection

Abstract: Single-stranded DNA with G-rich sequences can fold into secondary structures, G-quadruplexes, via intramolecular hydrogen-bonding interactions. This conformational change can be detected by a homogeneous assay method based on fluorescence resonance energy transfer (FRET) from a water-soluble cationic conjugated polymer (CCP) to a fluorescein chromophore labeled at the terminus of the G-quadruplex DNA. The space charge density around the DNA controls the efficiency of FRET from the CCP to the fluorescein. The higher FRET efficiency for the CCP/G-quadruplex pair is correlated to the stronger electrostatic interactions between the more condensed G-quadruplex and the CCP in comparison to the CCP/ssDNA pair. Since the potassium ion can specifically bind to the G-quadruplex DNA, the G-quartet-DNA/CCPs assembly can also be used as a platform to sense the potassium ion in water with high selectivity and sensitivity.

Structural changes involved in protein binding correlate with intrinsic motions of proteins in the unbound state

Abstract: Protein–protein binding usually involves structural changes that may extend beyond the rearrangements on a local scale, and cannot be explained by a classical lock-and-key mechanism. Several models have been advanced to explain the flexible binding of proteins such as the induced fit mechanism where the ligand is postulated to induce a conformational change at the interaction site upon binding, or the preexisting equilibrium hypothesis that assumes that protein samples an ensemble of conformations at equilibrium conditions and that the ligand binds selectively to an active conformation. We explored the equilibrium motions of proteins that exhibit relatively large (nonlocal) conformational changes upon protein binding using the Gaussian network model and the anisotropic network model of protein dynamics. For four complexes, LIR-1/HLA-A2, Actin/DNase I, CDK2/cyclin, and CDK6/p16INK4a, the motions calculated for the monomer exhibiting the largest conformational change, in its unbound (free) form, correlate with the experimentally observed structural changes upon binding. This study emphasizes the preexisting equilibrium/conformational selection as a mechanism for protein–protein interaction and lends support the concept that proteins, in their native conformation, are predisposed to undergo conformational fluctuations that are relevant to, or even required for, their biological functions.

Crosstalk in G protein-coupled receptors: Changes at the transmembrane homodimer interface determine activation

Abstract: Functional crosstalk between G protein-coupled receptors in a homo- or heterodimeric assembly likely involves conformational changes at the dimer interface, but the nature of this interface is not yet established, and the dynamic changes have not yet been identified. We have mapped the homodimer interface in the dopamine D2 receptor over the entire length of the fourth transmembrane segment (TM4) by crosslinking of substituted cysteines. Their susceptibilities to crosslinking are differentially altered by the presence of agonists and inverse agonists. The TM4 dimer interface in the inverse agonist-bound conformation is consistent with the dimer of the inactive form of rhodopsin modeled with constraints from atomic force microscopy. Crosslinking of a different set of cysteines in TM4 was slowed by inverse agonists and accelerated in the presence of agonists; crosslinking of the latter set locks the receptor in an active state. Thus, a conformational change at the TM4 dimer interface is part of the receptor activation mechanism.

Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering

Abstract: A family of genetically-encoded metabolite sensors has been constructed using bacterial periplasmic binding proteins (PBPs) linearly fused to protein fluorophores. The ligand-induced conformational change in a PBP allosterically regulates the relative distance and orientation of a fluorescence resonance energy transfer (FRET)-compatible protein pair. Ligand binding is transduced into a macroscopic FRET observable, providing a reagent for in vitro and in vivo ligand-measurement and visualization. Sensors with a higher FRET signal change are required to expand the dynamic range and allow visualization of subtle analyte changes under high noise conditions. Various observations suggest that factors other than inter-fluorophore separation contribute to FRET transfer efficiency and the resulting ligand-dependent spectral changes. Empirical and rational protein engineering leads to enhanced allosteric linkage between ligand binding and chromophore rearrangement; modifications predicted to decrease chromophore rotational averaging enhance the signal change, emphasizing the importance of the rotational freedom parameter kappa2 to FRET efficiency. Tighter allosteric linkage of the PBP and the fluorophores by linker truncation or by insertion of chromophores into the binding protein at rationally designed sites gave rise to sensors with improved signal change. High-response sensors were obtained with fluorescent proteins attached to the same binding PBP lobe, suggesting that indirect allosteric regulation during the hinge-bending motion is sufficient to give rise to a FRET response. The optimization of sensors for glucose and glutamate, ligands of great clinical interest, provides a general framework for the manipulation of ligand-dependent allosteric signal transduction mechanisms.

A Mechanism of Nonphotochemical Energy Dissipation, Independent from PsbS, Revealed by a Conformational Change in the Antenna Protein CP26

Abstract: The regulation of light harvesting in higher plant photosynthesis, defined as stress-dependent modulation of the ratio of energy transfer to the reaction centers versus heat dissipation, was studied by means of carotenoid biosynthesis mutants and recombinant light harvesting complexes (LHCs) with modified chromophore binding. The npq2 mutant of Arabidopsis thaliana, blocked in the biosynthesis of violaxanthin and thus accumulating zeaxanthin, was shown to have a lower fluorescence yield of chlorophyll in vivo and, correspondingly, a higher level of energy dissipation, with respect to the wild-type strain and npq1 mutant, the latter of which is incapable of zeaxanthin accumulation. Experiments on purified thylakoid membranes from all three mutants showed that the major source of the difference between the npq2 and wild-type preparations was a change in pigment to protein interactions, which can explain the lower chlorophyll fluorescence yield in the npq2 samples. Analysis of the xanthophyll binding LHC proteins showed that the Lhcb5 photosystem II subunit (also called CP26) undergoes a change in its pI upon binding of zeaxanthin. The same effect was observed in wild-type CP26 upon treatment that leads to the accumulation of zeaxanthin in the membrane and was interpreted as the consequence of a conformational change. This hypothesis was confirmed by the analysis of two recombinant proteins obtained by overexpression of the Lhcb5 apoprotein in Escherichia coli and reconstitution in vitro with either violaxanthin or zeaxanthin. The V and Z containing pigment-protein complexes obtained by this procedure showed different pIs and high and low fluorescence yields, respectively. These results confirm that LHC proteins exist in multiple conformations, an idea suggested by previous spectroscopic measurements (Moya et al., 2001), and imply that the switch between the different LHC protein conformations is activated by the binding of zeaxanthin to the allosteric site L2. The results suggest that the quenching process induced by the accumulation of zeaxanthin contributes to qI, a component of NPQ whose origin was previously poorly understood.

The initiation of antigen-induced B cell antigen receptor signaling viewed in living cells by fluorescence resonance energy transfer.

Abstract: Binding of antigen to the B cell antigen receptor (BCR) triggers signaling that ultimately leads to B cell activation. Using quantitative fluorescence resonance energy transfer imaging, we provide evidence here that the BCR is a monomer on the surface of resting cells. Binding of multivalent antigen clustered the BCR, resulting in the simultaneous phosphorylation of and a conformational change in the BCR cytoplasmic domains from a closed to an open form. Notably, the open conformation required immunoreceptor tyrosine-activation motif and continuous Src family kinase activity but not binding of the kinase Syk. Thus, the initiation of BCR signaling is a very dynamic process accompanied by reversible conformational changes induced by Src family kinase activity.

Optical sensors based on hybrid DNA/conjugated polymer complexes.

Abstract: Single-stranded DNA (ss-DNA) can specifically bind to various targets, including a complementary ss-DNA, ions, proteins, drugs, and so forth. When binding takes place, the oligonucleotide probe often undergoes a conformational transition. This conformational change of the negatively charged ss-DNA can be detected by using a water-soluble, cationic polythiophene derivative, which transduces the complex formation into an optical (colorimetric or fluorometric) signal without any labeling of the probe or the target. This simple and rapid methodology has enabled the specific and sensitive detection of nucleic acids and human thrombin. This new biophotonic tool can easily be applied to the detection of various other biomolecules and is also useful in the high-throughput screening of new drugs.

Loss of Bif-1 Suppresses Bax/Bak Conformational Change and Mitochondrial Apoptosis

Abstract: Bif-1, a member of the endophilin B protein family, interacts with Bax and promotes interleukin-3 withdrawal-induced Bax conformational change and apoptosis when overexpressed in FL5.12 cells. Here, we provide evidence that Bif-1 plays a regulatory role in apoptotic activation of not only Bax but also Bak and appears to be involved in suppression of tumorigenesis. Inhibition of endogenous Bif-1 expression in HeLa cells by RNA interference abrogated the conformational change of Bax and Bak, cytochrome c release, and caspase 3 activation induced by various intrinsic death signals. Similar results were obtained in Bif-1 knockout mouse embryonic fibroblasts. While Bif-1 did not directly interact with Bak, it heterodimerized with Bax on mitochondria in intact cells, and this interaction was enhanced by apoptosis induction and preceded the Bax conformational change. Moreover, suppression of Bif-1 expression was associated with an enhanced ability of HeLa cells to form colonies in soft agar and tumors in nude mice. Taken together, these findings support the notion that Bif-1 is an important component of the mitochondrial pathway for apoptosis as a novel Bax/Bak activator, and loss of this proapoptotic molecule may contribute to tumorigenesis.

Monitoring agonist-promoted conformational changes of β-arrestin in living cells by intramolecular BRET

Abstract: Recruitment of β-arrestin (β-arr) to agonist-stimulated G-protein-coupled receptors (GPCRs) has a crucial role in controlling signalling efficacy and selectivity. When translocated to the receptor, β-arr is believed to undergo important conformational rearrangement necessary for its downstream actions. To probe these changes in living cells, we constructed an intramolecular bioluminescence resonance energy transfer (BRET)-based biosensor, in which β-arr is sandwiched between the Renilla luciferase (Luc) and the yellow fluorescent protein (YFP). We show that the intramolecular BRET between Luc and YFP was significantly increased following GPCR activation, suggesting a conformational rearrangement bringing the amino terminus and carboxyl terminus of β-arr in closer proximity. Kinetic analysis showed that this conformational change follows the initial β-arr/receptor engagement. In addition to providing new insights into the agonist-induced conformational rearrangements of β-arr in living cells, the double-brilliance β-arr offers a universal biosensor for GPCR activation, allowing the study of native receptors in large-scale screening analysis.

Cellular prion protein inhibits proapoptotic Bax conformational change in human neurons and in breast carcinoma MCF-7 cells.

Abstract: Prion protein (PrP) prevents Bcl-2-associated protein X (Bax)-mediated cell death, but the step at which PrP inhibits is not known. We first show that PrP is very specific for Bax and cannot prevent Bak (Bcl-2 antagonist killer 1)-, tBid-, staurosporine- or thapsigargin-mediated cell death. As Bax activation involves Bax conformational change, mitochondrial translocation, cytochrome c release and caspase activation, we investigated which of these events was inhibited by PrP. PrP inhibits Bax conformational change, cytochrome c release and cell death in human primary neurons and MCF-7 cells. Serum deprivation-induced Bax conformational change is more rapid in PrP-null cells. PrP does not prevent active caspase-mediated cell death. PrP does not colocalize with Bax in normal or apoptotic primary neurons and cannot prevent Bax-mediated cytochrome c release in a mitochondrial cell-free system. We conclude that PrP protects against Bax-mediated cell death by preventing the Bax proapoptotic conformational change that occurs initially in Bax activation.

Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.

Abstract: Parathyroid hormone (PTH) and its related receptor (PTHR) are essential regulators of calcium homeostasis and bone physiology. PTH activates PTHR by interacting with a ligand-binding site localized within the N-terminal extracellular domain (the N-domain) and the domain comprising the seven transmembrane helices and the connecting extracellular loops (the J-domain). PTH binding triggers a conformational switch in the receptor, leading to receptor activation and subsequent cellular responses. The process of receptor activation occurs rapidly, within ≈1 s, but the binding event preceding receptor activation is not understood. By recording FRET between tetramethyl-rhodamine in PTH(1-34) and GFP in the N-domain of the receptor, we measured the binding event in real time in living cells. We show that the association time course between PTH(1-34) and PTHR involves a two-step binding process where the agonist initially binds the receptor with a fast time constant (τ ≈ 140 ms) and then with slower kinetics (τ ≈ 1 s). The fast and slow phases were assigned to hormone association to the receptor N- and J domains, respectively. Our data indicate that the slow binding step to the J-domain coincides with a conformational switch in the receptor, also monitored by FRET between the enhanced cyan fluorescent protein and the enhanced yellow fluorescent protein in the PTHR sensor, PTHR enhanced cyan fluorescent protein/enhanced yellow fluorescent protein (PTHRCFP/YFP). These data suggest that the conformational change that switches the receptor into its active state proceeds in a sequential manner, with the first rapid binding step event preceding receptor activation by PTH(1-34).

Conformational Changes in Protein Loops and Helices Induced by Post-Translational Phosphorylation

Abstract: Post-translational phosphorylation is a ubiquitous mechanism for modulating protein activity and protein-protein interactions. In this work, we examine how phosphorylation can modulate the conformation of a protein by changing the energy landscape. We present a molecular mechanics method in which we phosphorylate proteins in silico and then predict how the conformation of the protein will change in response to phosphorylation. We apply this method to a test set comprised of proteins with both phosphorylated and non-phosphorylated crystal structures, and demonstrate that it is possible to predict localized phosphorylation-induced conformational changes, or the absence of conformational changes, with near-atomic accuracy in most cases. Examples of proteins used for testing our methods include kinases and prokaryotic response regulators. Through a detailed case study of cyclin-dependent kinase 2, we also illustrate how the computational methods can be used to provide new understanding of how phosphorylation drives conformational change, why substituting Glu or Asp for a phosphorylated amino acid does not always mimic the effects of phosphorylation, and how a phosphatase can “capture” a phosphorylated amino acid. This work illustrates how computational methods can be used to elucidate principles and mechanisms of post-translational phosphorylation, which can ultimately help to bridge the gap between the number of known sites of phosphorylation and the number of structures of phosphorylated proteins.

Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor.

Abstract: Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen-binding domain (PABD) and a calmodulin (CaM)-activated adenylyl cyclase domain. Here, we report the crystal structures of CaM-bound EF, revealing the architecture of EF PABD. CaM has N- and C-terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF–CaM complex show how EF locks the N-terminal domain of CaM into a closed conformation regardless of its calcium-loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM and uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF–CaM complexed with nucleotides show that EF uses two-metal–ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3′OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two-metal–ion catalysis, suggesting the catalytic mechanism-driven convergent evolution of two structurally diverse adenylyl cyclases.

T cell receptor engagement by peptide–MHC ligands induces a conformational change in the CD3 complex of thymocytes

Abstract: The T cell receptor (TCR) can recognize a variety of cognate peptide/major histocompatibility complex (pMHC) ligands and translate their affinity into distinct cellular responses. To achieve this, the nonsignaling αβ heterodimer communicates ligand recognition to the CD3 signaling subunits by an unknown mechanism. In thymocytes, we found that both positive- and negative-selecting pMHC ligands expose a cryptic epitope in the CD3 complex upon TCR engagement. This conformational change is induced in vivo and requires the expression of cognate MHC. We conclude that TCR engagement with a cognate pMHC ligand induces a conformational change in the CD3 complex of thymocytes and propose that this marks an initial event during thymic selection that signals the recognition of self-antigen.

PIP2 activates TRPV5 and releases its inhibition by intracellular Mg2

Abstract: The transient receptor potential type V5 channel (TRPV5) is a Ca2+-selective TRP channel important for epithelial Ca2+ transport. Intracellular Mg2+ causes a fast voltage-dependent block of the TRPV5 channel by binding to the selectivity filter. Here, we report that intracellular Mg2+ binding to the selectivity filter of TRPV5 also causes a slower reversible conformational change leading to channel closure. We further report that PIP2 activates TRPV5. Activation of TRPV5 by PIP2 is independent of Mg2+. Yet, PIP2 decreases sensitivity of the channel to the Mg2+-induced slow inhibition. Mutation of aspartate-542, a critical Mg2+-binding site in the selectivity filter, abolishes Mg2+-induced slow inhibition. PIP2 has no effects on Mg2+-induced voltage-dependent block. Thus, PIP2 prevents the Mg2+-induced conformational change without affecting Mg2+ binding to the selectivity filter. Hydrolysis of PIP2 via receptor activation of phospholipase C sensitizes TRPV5 to the Mg2+-induced slow inhibition. These results provide a novel mechanism for regulation of TRP channels by phospholipase C-activating hormones via alteration of the sensitivity to intracellular Mg2+.

Crystal structure and functional analysis of DEAD-box protein Dhh1p.

Abstract: The control of mRNA translation and degradation are critical for proper gene expression. A key regulator of both translation and degradation is Dhh1p, which is a DEAD-box protein, and functions both to repress translation and enhance decapping. We describe the crystal structure of the N- and C-terminal truncated Dhh1p (tDhh1p) determined at 2.1 A resolution. This reveals that, like other DEAD-box proteins, tDhh1p contains two RecA-like domains, although with a unique arrangement. In contrast to eIF4A and mjDEAD, in which no motif interactions exist, in Dhh1p, motif V interacts with motif I and the Q-motif, thereby linking the two domains together. Electrostatic potential mapping combined with mutagenesis reveals that motifs I, V, and VI are involved in RNA binding. In addition, trypsin digestion of tDhh1p suggests that ATP binding enhances an RNA-induced conformational change. Interestingly, some mutations located in the conserved motifs and at the interface between the two Dhh1 domains confer dominant negative phenotypes in vivo and disrupt the conformational switch in vitro. This suggests that this conformational change is required in Dhh1 function and identifies key residues involved in that transition.

Simple energy landscape model for the kinetics of functional transitions in proteins.

Abstract: It is evident that protein conformational transitions play important roles in biological machinery; however, detailed pictures of these transition processes capable of making kinetic prediction are not yet available. For a full description of these transitions, we first need to describe kinematically movements between stable states. Then, more importantly, a free energy profile associated with the conformational change needs to be obtained. Recently, a new model to describe the energy landscape of protein conformational changes was applied to the conformational transition of adenylate kinase [Miyashita, O.; Onuchic, J. N.; Wolynes, P. G. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12570−12575]. In this model, the conformational change coupled to the ligand binding is described as a switching between two energy surfaces that correspond to ligand bound and unbound states. The nonlinearity of the protein conformational changes is described through an iterative usage of normal mode calculations. In addition, ano...

Movements of the ε‐subunit during catalysis and activation in single membrane‐bound H+‐ATP synthase

Abstract: F0F1-ATP synthases catalyze proton transport-coupled ATP synthesis in bacteria, chloroplasts, and mitochondria. In these complexes, the epsilon-subunit is involved in the catalytic reaction and the activation of the enzyme. Fluorescence-labeled F0F1 from Escherichia coli was incorporated into liposomes. Single-molecule fluorescence resonance energy transfer (FRET) revealed that the epsilon-subunit rotates stepwise showing three distinct distances to the b-subunits in the peripheral stalk. Rotation occurred in opposite directions during ATP synthesis and hydrolysis. Analysis of the dwell times of each FRET state revealed different reactivities of the three catalytic sites that depended on the relative orientation of epsilon during rotation. Proton transport through the enzyme in the absence of nucleotides led to conformational changes of epsilon. When the enzyme was inactive (i.e. in the absence of substrates or without membrane energization), three distances were found again, which differed from those of the active enzyme. The three states of the inactive enzyme were unequally populated. We conclude that the active-inactive transition was associated with a conformational change of epsilon within the central stalk.

Direct correlation between adsorption-induced changes in protein structure and platelet adhesion.

Abstract: It is widely recognized that adsorbed proteins on biomaterial surfaces tend to initiate thrombus formation, although the specific mechanisms involved are still not well understood. In attempts to decrease the conformational change of adsorbed proteins, surface treatments that reduce surface hydrophobicity have been considered, such as the sulfonation of low-density polyethylene and isotactic polypropylene. The objectives of this present research were to study how changes in surface chemistry influence the degree of conformational change of adsorbing proteins and to investigate the correlation between the change in adsorbed protein structure and platelet response. Adsorbed porcine serum albumin and porcine fibrinogen were used as the model proteins for determining the effects of sulfonation on protein conformational change. Circular dichroism spectroscopy studies showed that the proteins were less altered structurally on the sulfonated surfaces. Platelet adhesion studies were used to correlate the number of adhered platelets with the amount of conformational change in adsorbed proteins on the polymer surface. The results of these studies show a linear correlation between platelet adhesion and the degree of adsorption-induced protein conformational change. These findings suggest that the degree of protein conformational change after adsorption is a dominant mechanism governing platelet interactions with biomaterial surfaces.

Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase.

Abstract: Ensemble kinetics and single-molecule fluorescence microscopy were used to study conformational transitions associated with enzyme catalysis by dihydrofolate reductase (DHFR). The active site loop of DHFR was labeled with a fluorescence quencher, QSY35, at amino acid position 17, and the fluorescent probe, Alexa555, at amino acid 37, by introducing cysteines at these sites with site-specific mutagenesis. The distance between the probes was such that approximately 50% fluorescence resonance energy transfer (FRET) occurred. The double-labeled enzyme retained essentially full catalytic activity, and stopped-flow studies of both the forward and reverse reactions revealed that the distance between probes increased prior to hydride transfer. A fluctuation in fluorescence intensity of single molecules of DHFR was observed in an equilibrium mixture of substrates but not in their absence. Ensemble rate constants were derived from the distributions of lifetimes observed and attributed to a reversible conformational change. Studies were carried out with both NADPH and NADPD as substrates, with no measurable isotope effect. Similar studies with a G121V mutant DHFR resulted in smaller rate constants. This mutant DHFR has reduced catalytic activity, so that the collective data for the conformational change suggest that the conformational change being observed is associated with catalysis and probably represents a conformational change prior to hydride transfer. If the change in fluorescence is attributed to a change in FRET, the distance change associated with the conformational change is approximately 1-2 A. These results are correlated with other measurements related to conformation coupled catalysis.