Showing papers on "Conformational change published in 2016"
TL;DR: Fluorescence spectroscopy on a series of aqueous solutions of poly(acrylic acid) containing a luminescent label showed that polymers with molar mass, Mn < 16.5 kDa did not exhibit a pH responsive conformational change, which is typical of higher molarMass poly(Acrylic acid), while large molarmass polymers did exhibit pH-dependent diffusion.
Abstract: Fluorescence spectroscopy on a series of aqueous solutions of poly(acrylic acid) containing a luminescent label showed that polymers with molar mass, Mn < 16.5 kDa did not exhibit a pH responsive conformational change, which is typical of higher molar mass poly(acrylic acid). Below this molar mass, polymers remained in an extended conformation, regardless of pH. Above this molar mass, a pH-dependent conformational change was observed. Diffusion-ordered nuclear magnetic resonance spectroscopy confirmed that low molar mass polymers did not undergo a conformational transition, although large molar mass polymers did exhibit pH-dependent diffusion.
278 citations
TL;DR: The design, synthesis, and conformational behavior of photoresponsive foldamers bound in a phospholipid bilayer akin to a biological membrane phase are reported, pointing the way toward development of small-molecule–based switches in membrane environments.
Abstract: The dynamic properties of foldamers, synthetic molecules that mimic folded biomolecules, have mainly been explored in free solution. We report on the design, synthesis, and conformational behavior of photoresponsive foldamers bound in a phospholipid bilayer akin to a biological membrane phase. These molecules contain a chromophore, which can be switched between two configurations by different wavelengths of light, attached to a helical synthetic peptide that both promotes membrane insertion and communicates conformational change along its length. Light-induced structural changes in the chromophore are translated into global conformational changes, which are detected by monitoring the solid-state (19)F nuclear magnetic resonance signals of a remote fluorine-containing residue located 1 to 2 nanometers away. The behavior of the foldamers in the membrane phase is similar to that of analogous compounds in organic solvents.
130 citations
TL;DR: It is shown that conformational changes happening upon adsorption of myoglobin and BSA are dependent on the size of the nanoparticle they are adsorbing to, and the plausibility of commonly used explanations for the phenomenon of nanoparticle size‐dependent conformational change is evaluated.
Abstract: The use of nanomaterials in bioapplications demands a detailed understanding of protein–nanoparticle interactions. Proteins can undergo conformational changes while adsorbing onto nanoparticles, but studies on the impact of particle size on conformational changes are scarce. We have shown that conformational changes happening upon adsorption of myoglobin and BSA are dependent on the size of the nanoparticle they are adsorbing to. Out of eight initially investigated model proteins, two (BSA and myoglobin) showed conformational changes, and in both cases this conformational change was dependent on the size of the nanoparticle. Nanoparticle sizes ranged from 30 to 1000 nm and, in contrast to previous studies, we attempted to use a continuous progression of sizes in the range found in live viruses, which is an interesting size of nanoparticles for the potential use as drug delivery vehicles. Conformational changes were only visible for particles of 200 nm and bigger. Using an optimized circular dichroism protocol allowed us to follow this conformational change with regard to the nanoparticle size and, thanks to the excellent temporal resolution also in time. We uncovered significant differences between the unfolding kinetics of myoglobin and BSA. In this study, we also evaluated the plausibility of commonly used explanations for the phenomenon of nanoparticle size‐dependent conformational change. Currently proposed mechanisms are mostly based on studies done with relatively small particles, and fall short in explaining the behavior seen in our studies.
122 citations
01 Jan 2016
TL;DR: Leucine-enkephalin appears to have the same conformation as its methionine homolog, consistent with published nuclear magnetic resonance parameters--coupling constants, temperature dependence of the chemical shift, and spin-lattice relaxation times.
Abstract: Low-energy conformations of methionine- enkephalin were generated by means of an empirical method of computation. Many compact conformations, including those containing various standard bends, were of comparable energy. However, one conformation was found to have a potential en- ergy about 5 kcal/mol (21 X 103 J/mol) below that of the large group of compact conformations. In this conformation, the 3- glycyl and 4-phenylalanyl residues form a bend of type II'. The conformation is stabilized by a hydrogen bond between the OH group of the 1-tyrosine side chain and the C=O group of 3-gly- cine or 4-phenylalanine. The phenylalanine and methionine side chains are relatively unrestricted. The conformation is consis- tent with published nuclear magnetic resonance parameters- coupling constants, temperature dependence of the chemical shift, and spin-lattice relaxation times. It is likely that the mol- ecule undergoes a conformational change when it is bound to the receptor. Leucine-enkephalin appears to have the same conformation as its methionine homolog.
112 citations
TL;DR: Cav1 phosphorylation destabilizes plasma membrane–associated Cav-1 oligomers and thereby is crucial for regulating the fission of caveolae from the plasma membrane in vascular endothelial cells.
Abstract: Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane
90 citations
TL;DR: Morin was found to exert significant inhibition activity on α-glucosidase in a reversible mixed-type manner with an IC50 value of (4.48 ± 0.04) μM and was also found to exhibit inhibition in the generation of advanced glycation end products which was related to the long term complications of diabetes.
Abstract: It is important to investigate the inhibition of α-glucosidase due to its correlation with type 2 diabetes. Morin was found to exert significant inhibition activity on α-glucosidase in a reversible mixed-type manner with an IC50 value of (4.48 ± 0.04) μM. Analyses of fluorescence and circular dichroism spectra indicated that the formation of the morin-α-glucosidase complex was driven mainly by hydrophobic forces and hydrogen bonding, and caused the conformational changes of α-glucosidase. The phase diagrams of fluorescence showed that the conformational change process was monophasic without intermediates. Molecular docking indicated that morin mainly interacted with amino acid residues located close to the active site of α-glucosidase, which may move to cover the active pocket to reduce the binding of the substrate and then inhibit the catalytic activity. Morin was also found to exhibit inhibition in the generation of advanced glycation end products which was related to the long term complications of diabetes.
82 citations
TL;DR: General results for the chemical relaxation rates of conformational-selection and induced-fit binding processes that hold for all concentrations of proteins and ligands and, thus, go beyond the standard pseudo-first-order approximation of large ligand concentration are presented.
Abstract: Protein binding often involves conformational changes. Important questions are whether a conformational change occurs prior to a binding event (‘conformational selection’) or after a binding event (‘induced fit’), and how conformational transition rates can be obtained from experiments. In this article, we present general results for the chemical relaxation rates of conformational-selection and induced-fit binding processes that hold for all concentrations of proteins and ligands and, thus, go beyond the standard pseudo-first-order approximation of large ligand concentration. These results allow to distinguish conformational-selection from induced-fit processes—also in cases in which such a distinction is not possible under pseudo-first-order conditions—and to extract conformational transition rates of proteins from chemical relaxation data.
71 citations
TL;DR: Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na+ and transmembrane helices 1 and 8, whereas Na+ binding at Na1 influences conformational change through a network of intermediary interactions.
64 citations
TL;DR: This work reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM) and lengthened the reversion half-life of iLIDs.
Abstract: Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the “off” state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the “on” state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5–100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-...
64 citations
TL;DR: The assessment of conformational changes associated with ligand binding by ITC is discussed, and the “conformational selection” and “induced fit” models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins.
60 citations
TL;DR: It is found that both conformational selection and induced fit play a role in the binding mechanism, reconciling opposing views held in the literature.
Abstract: Understanding the conformational changes associated with the binding of small ligands to their biological targets is a fascinating and meaningful question in chemistry, biology and drug discovery. One of the most studied and important is the so-called “DFG-flip” of tyrosine kinases. The conserved three amino-acid DFG motif undergoes an “in to out” movement resulting in a particular inactive conformation to which “type II” kinase inhibitors, such as the anti-cancer drug Imatinib, bind. Despite many studies, the details of this prototypical conformational change are still debated. Here we combine various NMR experiments and surface plasmon resonance with enhanced sampling molecular dynamics simulations to shed light into the conformational dynamics associated with the binding of Imatinib to the proto-oncogene c-Src. We find that both conformational selection and induced fit play a role in the binding mechanism, reconciling opposing views held in the literature. Moreover, an external binding pose and local unfolding (cracking) of the aG helix are observed.
TL;DR: This work exploits the fact that outer membrane proteins usually lack reactive cysteines and that paramagnetic spin labels entering the periplasm are selectively reduced to achieve specific labeling of the cobalamin transporter BtuB in Escherichia coli to demonstrate the first proof of concept for the use of PELDOR to observe conformational changes in a membrane protein in intact cells.
Abstract: An unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. Pulsed electron–electron double resonance (PELDOR) is a well-established technique to follow conformational changes in purified membrane protein complexes. Here we demonstrate the first proof of concept for the use of PELDOR to observe conformational changes in a membrane protein in intact cells. We exploit the fact that outer membrane proteins usually lack reactive cysteines and that paramagnetic spin labels entering the periplasm are selectively reduced to achieve specific labeling of the cobalamin transporter BtuB in Escherichia coli. We characterize conformational changes in the second extracellular loop of BtuB upon ligand binding and compare the PELDOR data with high-resolution crystal structures. Our approach avoids detergent extraction, purification, and reconstitution usually required for these systems. With this approach, s...
TL;DR: The functional impact of two natural anemia-causing glycine-to-arginine mutations that impaired transition metal transport in both human Nramp2 and DraNramp are investigated.
TL;DR: The detailed molecular view of the binding site paves the way for the design of a new generation of selectin antagonists, of special interest, since their therapeutic potential was recently demonstrated with the pan-selectin antagonists GMI-1070 (Rivipansel).
Abstract: E-selectin is a cell-adhesion molecule of the vascular endothelium that promotes essential leukocyte rolling in the early inflammatory response by binding to glycoproteins containing the tetrasaccharide sialyl Lewis(x) (sLe(x)). Efficient leukocyte recruitment under vascular flow conditions depends on an increased lifetime of E-selectin/ligand complexes under tensile force in a so-called catch-bond binding mode. Co-crystal structures of a representative fragment of the extracellular E-selectin region with sLe(x) and a glycomimetic antagonist thereof reveal an extended E-selectin conformation, which is identified as a high-affinity binding state of E-selectin by molecular dynamics simulations. Small-angle X-ray scattering experiments demonstrate a direct link between ligand binding and E-selectin conformational transition under static conditions in solution. This permits tracing a series of concerted structural changes connecting ligand binding to conformational stretching as the structural basis of E-selectin catch-bond-mediated leukocyte recruitment. The detailed molecular view of the binding site paves the way for the design of a new generation of selectin antagonists. This is of special interest, since their therapeutic potential was recently demonstrated with the pan-selectin antagonists GMI-1070 (Rivipansel).
TL;DR: A model by which W-acidic cargo binding regulates the activity of the holoenzyme is proposed, describing how a previously unnoticed intramolecular interaction between the KLC2TPR domain and a highly conserved peptide motif within an unstructured region of the molecule occludes a key cargo binding site on the light-chain TPR domain.
Abstract: The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLC(TPR)), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2(TPR) domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme.
TL;DR: Substantial results indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation.
Abstract: The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott–Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a “short-pitch” conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP’s CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP–Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3′s barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex.
TL;DR: A 2.6-Å-resolution crystal structure of a heterotrimer of fission yeast Dcp2, its essential activator Dcp1, and the human NMD cofactor PNRC2, in complex with a tight-binding cap analog reveals that coactivators promote RNA binding and the catalytic step of decapping, possibly through different conformational states.
Abstract: Removal of the 5' cap on mRNA by the decapping enzyme Dcp2 is a critical step in 5'-to-3' mRNA decay. Understanding the structural basis of Dcp2 activity has been a challenge because Dcp2 is dynamic and has weak affinity for the cap substrate. Here we present a 2.6-A-resolution crystal structure of a heterotrimer of fission yeast Dcp2, its essential activator Dcp1, and the human NMD cofactor PNRC2, in complex with a tight-binding cap analog. Cap binding is accompanied by a conformational change in Dcp2, thereby forming a composite nucleotide-binding site comprising conserved residues in the catalytic and regulatory domains. Kinetic analysis of PNRC2 revealed that a conserved short linear motif enhances both substrate affinity and the catalytic step of decapping. These findings explain why Dcp2 requires a conformational change for efficient catalysis and reveals that coactivators promote RNA binding and the catalytic step of decapping, possibly through different conformational states.
TL;DR: It is shown that conformational signaling, although thus far underappreciated, is a general and robust signaling principle that most of the time operates in close interplay with covalent signals in the cell.
Abstract: Signal transduction is the primary process by which cells respond to changes in their physical and chemical environments. Cellular response is initiated through a signaling protein (a receptor), which interacts with the “signal”, most often a novel molecule outside or inside the cell. The mechanism of activation of the receptor is a conformational change and/or covalent modification, which then sets in motion a signaling pathway, i.e. a cascade of modification and binding events that relay and amplify the message to eventually alter the state of the cell. In reflection of this general perception, concepts such as the “second messenger” and the “phosphorylation cascade” dominate our views of signal transduction. The idea I advocate here is that the non-covalent change in protein conformation itself might serve as the initial or intermittent “signal” in the cascade, and it is often the primary event being recognized and interpreted by downstream receptor(s). This signaling principle is intertwined with many other cellular regulatory concepts, such as (pathway) allostery, conformational spread, induced folding/unfolding, conformational memory, the hierarchical assembly of complexes, and the action of regulatory chaperones and prions. By elaborating on many examples and also recent advances in experimental methodology, I show that conformational signaling, although thus far underappreciated, is a general and robust signaling principle that most of the time operates in close interplay with covalent signals in the cell.
TL;DR: The paper provides a novel insight into the conformational changes in G-quadruplex and presents an efficient step to resolve the challenging problem about Pb2+ detection in Na+-containing real samples.
Abstract: Here, we first find that Na+ can induce Pb2+-stabilized T30695 undergoing conformational transition from partly parallel to completely parallel, and further forming a dimeric G-quadruplex, which was characterized by circular dichroism (CD) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and native polyacrylamide gel electrophoresis (PAGE). Thermal denaturation experiments show that the transforming process is a thermodynamics-driven process. Furthermore, the presence of Na+ further improves the binding efficiency of Pb2+-stabilized T30695 with the fluorescent probe (such as ZnPPIX). Based on the fact, with a partially hybridized double-stranded DNA (ds-DNA) containing T30695 as a sensing probe and ZnPPIX as a fluorescence probe, the effect of Na+ on Pb2+ detection is subsequently investigated. The presence of Na+ (varied from 0.3 mM to 500 mM) simultaneously increases the read-out and background fluorescence, which results in a decreased signal-to...
TL;DR: It is demonstrated how the isoenergetic energy landscape of higher-order coiled coils can enable the formation of an oligomerization switch by insertion of a single destabilizing element into an otherwise stable computationally designed scaffold.
TL;DR: Two conformations of complexin-1 bound to the ternary SNARE complex may be involved in activation of synchronous neurotransmitter release, whereas both conformations may beinvolved in regulating spontaneous release.
Abstract: Complexin regulates spontaneous and activates Ca(2+)-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of complexin-1 bound to the ternary SNARE complex. In the cis conformation, complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of complexin-1. The complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release.
TL;DR: The crystal structure of yeast V1 presented here shows that activity silencing involves a large conformational change of subunit H, with its C‐terminal domain rotating ~150° from a position near the membrane in holo V‐ATPase to a position at the bottom of V1 near an open catalytic site.
Abstract: Vacuolar ATPases (V-ATPases) are essential proton pumps that acidify the lumen of subcellular organelles in all eukaryotic cells and the extracellular space in some tissues. V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, wherein the soluble catalytic sector, V1, is released from the membrane and its MgATPase activity silenced. The crystal structure of yeast V1 presented here shows that activity silencing involves a large conformational change of subunit H, with its C-terminal domain rotating ~150° from a position near the membrane in holo V-ATPase to a position at the bottom of V1 near an open catalytic site. Together with biochemical data, the structure supports a mechanistic model wherein subunit H inhibits ATPase activity by stabilizing an open catalytic site that results in tight binding of inhibitory ADP at another site.
TL;DR: In this paper, the authors employed enhanced sampling atomistic simulations to reveal DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215.
Abstract: Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling.
TL;DR: A new conformation of the E. coli RNR pathway residue 731 was trapped during long-range radical transfer across the αβ subunit interface.
Abstract: Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all living organisms. The catalytic cycle of E. coli RNR involves a long-range proton-coupled electron transfer (PCET) from a tyrosyl radical (Y122˙) in subunit β2 to a cysteine (C439) in the active site of subunit α2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 A and involves a specific pathway of redox active amino acids (Y122 ↔ [W48?] ↔ Y356 in β2 to Y731 ↔ Y730 ↔ C439 in α2). The mechanisms of the PCET steps at the interface of the α2β2 complex remain puzzling due to a lack of structural information for this region. Recently, DFT calculations on the 3-aminotyrosyl radical (NH2Y731˙)-α2 trapped by incubation of NH2Y731-α2/β2/CDP(substrate)/ATP(allosteric effector) suggested that R411-α2, a residue close to the α2β2 interface, interacts with NH2Y731˙ and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH2Y731-α2 with a R411A substitution. NH2Y731˙/R411A generated upon incubation of NH2Y731/R411A-α2/β2/CDP/ATP was investigated using multi-frequency (34, 94 and 263 GHz) EPR, 34 GHz pulsed electron-electron double resonance (PELDOR) and electron-nuclear double resonance (ENDOR) spectroscopies. The data indicate a large conformational change in NH2Y731˙/R411A relative to the NH2Y731˙ single mutant. Particularly, the inter-spin distance from NH2Y731˙/R411A in one αβ pair to Y122˙ in a second αβ pair decreases by 3 A in the presence of the R411A mutation. This is the first experimental evidence for the flexibility of pathway residue Y731-α2 in an α2β2 complex and suggests a role for R411 in the stacked Y731/Y730 conformation involved in collinear PCET. Furthermore, NH2Y731˙/R411A serves as a probe of the PCET process across the subunit interface.
TL;DR: The results suggest that activation proceeds via a conformational change in the PBP induced by lipoprotein binding, and indicates that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo.
Abstract: To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.
TL;DR: P pH-triggered conformational structure of the ELP linker shifts RGD-mediated cancer cell targeting from non-active to active, indicating that the reversible shift in ELP secondary structure may be used to engineer targeted drug delivery vehicles with tunable uptake.
TL;DR: The effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL, onto the F or G helices and using DEER to measure the distance between labels.
Abstract: In this study, the effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL (bifunctional spin-label) onto the F or G helices and using DEER (double electron-electron resonance) to measure the distance between labels. Recent EPR and crystallographic studies have observed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state. However, this change was not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound P450cam (Fe(2+)CO). In addition, recent NMR studies have failed to observe a change in P450cam conformation upon binding Pdx. Hence, resolving these issues is important for a full understanding the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformation when labeled on either the F or G helices with MTSL. BSL at these sites can either narrow the distance distribution widths dramatically or alter the extent of the conformational change. In addition, we report DEER spectra on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed. This indicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted by Pdx binding. Finally, we report the open form P450cam crystal structure with substrate bound, which suggests that crystal packing effects may prevent conformational conversion. Using multiple labeling approaches, DEER provides a unique perspective to resolve how the conformation of P450cam depends on Pdx and ligand states.
TL;DR: A peptidomimetic compound undergoes a reversible single-crystal-to-single-Crystal transformation upon guest release/uptake with the transformation involving a drastic conformational change, resulting in an unprecedented "CH-π zipper" which can reversibly open and close (through the formation of CH-π interactions), thus allowing for guest sensing.
Abstract: A peptidomimetic compound undergoes a reversible single-crystal-to-single-crystal transformation upon guest release/uptake with the transformation involving a drastic conformational change. The extensive and reversible alteration in the solid state is connected to the formation of an unprecedented "CH-π zipper" which can reversibly open and close (through the formation of CH-π interactions), thus allowing for guest sensing.
TL;DR: The results strongly indicate that anti-inflammatory effects in TNF-α-stimulated endothelial cells by acetylation are tightly linked to secreted APE1/Ref-1, which inhibits T NF-α binding to TNFR1 by reductive conformational change, with suggestion as an endogenous inhibitor of vascular inflammation.
Abstract: Apurinic apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is a multifunctional protein with redox activity and is proved to be secreted from stimulated cells. The aim of this study was to evaluate the functions of extracellular APE1/Ref-1 with respect to leading anti-inflammatory signaling in TNF-α-stimulated endothelial cells in response to acetylation. Treatment of TNF-α-stimulated endothelial cells with an inhibitor of deacetylase that causes intracellular acetylation, considerably suppressed vascular cell adhesion molecule-1 (VCAM-1). During TSA-mediated acetylation in culture, a time-dependent increase in secreted APE1/Ref-1 was confirmed. The acetyl moiety of acetylated-APE1/Ref-1 was rapidly removed based on the removal kinetics. Additionally, recombinant human (rh) APE1/Ref-1 with reducing activity induced a conformational change in rh TNF-α receptor 1 (TNFR1) by thiol-disulfide exchange. Following treatment with the neutralizing anti-APE1/Ref-1 antibody, inflammatory signals via the binding of TNF-α to TNFR1 were remarkably recovered, leading to up-regulation of reactive oxygen species generation and VCAM-1, in accordance with the activation of p66shc and p38 MAPK. These results strongly indicate that anti-inflammatory effects in TNF-α-stimulated endothelial cells by acetylation are tightly linked to secreted APE1/Ref-1, which inhibits TNF-α binding to TNFR1 by reductive conformational change, with suggestion as an endogenous inhibitor of vascular inflammation.
TL;DR: PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis, and the resultant increased thermal and proteolytic stability of HSA may provide greater longevity to H SA in plasma.
Abstract: Phycocyanobilin (PCB) binds with high affinity (2.2 x 106 M-1 at 25°C) to human serum albumin (HSA) at sites located in IB and IIA subdomains. The aim of this study was to examine effects of PCB binding on protein conformation and stability. Using 300 ns molecular dynamics (MD) simulations, UV-VIS spectrophotometry, CD, FT-IR, spectrofluorimetry, thermal denaturation and susceptibility to trypsin digestion, we studied the effects of PCB binding on the stability and rigidity of HSA, as well as the conformational changes in PCB itself upon binding to the protein. MD simulation results demonstrated that HSA with PCB bound at any of the two sites showed greater rigidity and lower overall and individual domain flexibility compared to free HSA. Experimental data demonstrated an increase in the α-helical content of the protein and thermal and proteolytic stability upon ligand binding. PCB bound to HSA undergoes a conformational change to a more elongated conformation in the binding pockets of HSA. PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis. The results from this work explain at molecular level, conformational changes and stabilization of HSA structure upon ligand binding. The resultant increased thermal and proteolytic stability of HSA may provide greater longevity to HSA in plasma.