Role Of Conformational Changes Research Articles

Conformational and Solvent Effects on the Photoinduced Electron Transfer Dynamics of a Zinc Phthalocyanine-Benzoperylenetriimide Conjugate: A Nonadiabatic Dynamics Simulation.

Herein, we employed a combination of static electronic structure calculations and nonadiabatic dynamics simulations at linear-response time dependent density functional theory (LR-TDDFT) level with the optimally tuned range-separated hybrid (OT-RSH) functional to explore the ultrafast photoinduced dynamics of a zinc phthalocyanine-benzoperylenetriimide (ZnPc-BPTI) conjugate. Due to the flexibility of the linker, we identified two major conformations: the stacked conformation (ZnPc-BPTI-1) and the extended conformation (ZnPc-BPTI-2). Since the charge transfer states are much lower than the lowest local excitation in ZnPc-BPTI-1, which is contrary to ZnPc-BPTI-2, the ultrafast electron transfer (~3.6 ps) is only observed in the nonadiabatic simulations of ZnPc-BPTI-1 upon local excitation around the absorption maximum of ZnPc. However, when considering the solvent effects in benzonitrile: the lowest S1 states are both charge transfer states from ZnPc to BPTI for different conformers. Subsequent nonadiabatic dynamics simulations indicate that both conformers experience ultrafast electron transfer in benzonitrile with two time constants of 90 [100] fs and 1.40 [1.43] ps. Our present work not only agrees well with previous experimental study, but also points out the important role of conformational changes and solvent effects in regulating the photodynamics of organic donor-acceptor conjugates.

A role for conformational changes in enzyme catalysis

The role played by conformational changes in enzyme catalysis is controversial. In addition to examining specific enzymes, studying formal models can help identify the conditions under which conformational changes promote catalysis. Here, we present a model demonstrating how conformational changes can break a generic trade-off due to the conflicting requirements of successive steps in catalytic cycles, namely high specificity for the transition state to accelerate the chemical transformation and low affinity for the products to favor their release. The mechanism by which the trade-off is broken is a transition between conformations with different affinities for the substrate. The role of the effector that induces the transition is played by a substrate “handle,” a part of the substrate that is not chemically transformed but whose interaction with the enzyme is nevertheless essential to rapidly complete the catalytic cycle. A key element of the model is the formalization of the constraints causing the trade-off that the presence of multiple states breaks, which we attribute to the strong chemical similarity between successive reaction states—substrates, transition states, and products. For the sake of clarity, we present our model for irreversible one-step unimolecular reactions. In this context, we demonstrate how the different forms that chemical similarities between reaction states can take impose limits on the overall catalytic turnover. We first analyze catalysts without internal degrees of freedom and then show how two-state catalysts can overcome their limitations. Our results recapitulate previous proposals concerning the role of conformational changes and substrate handles in a formalism that makes explicit the constraints that elicit these features. In addition, our approach establishes links with studies in the field of heterogeneous catalysis, where the same trade-offs are observed and where overcoming them is a well-recognized challenge.

Binding of Selected Aroma Compounds to Myofibrillar Protein, Sarcoplasmic Protein, and Collagen during Thermal Treatment: Role of Conformational Changes and Degradation of Proteins.

To investigate the effects of conformational changes and thermal degradation of myofibrillar protein (MP), sarcoplasmic protein (SP), and collagen (CO) on the binding ability for aroma compounds during heating. Using SDS-PAGE, HPLC, and LC-MS/MS, a consistent rise in the total concentration of peptides and free amino acids formed by the thermal degradation of proteins was observed. The surface hydrophobicity, total sulfhydryl content, particle size, and secondary structure content of proteins changed significantly over time. Furthermore, the aroma binding ability of proteins was determined by gas chromatography-mass spectrometry. The results revealed an increase in binding ability during 5 or 10 min of heating due to protein unfolding and the accumulation of degradation products. However, the binding ability decreased due to protein aggregation with prolonged heating. Notably, all proteins exhibited strong affinity toward (E)-2-octenal, (E,E)-2,4-decadienal, 2-methyl-3-furanthiol, and dimethyl trisulfide. The binding ability of MP and SP was similar but differed significantly from that of CO, which had lower binding ability for hexanal, (E)-2-octenal, (E,E)-2,4-decadienal, and dimethyl trisulfide compared to MP and SP.

Role of Conformational Changes in DNA Recognition in Transcription Factors having Helix-Turn-Helix Motif

DNA-binding proteins play a crucial role in various cellular processes, some of these have a unique structural arrangement, the helix-turn-helix motif, which enables them to bind to DNA molecules in a specific manner. Upon binding to DNA, conformational changes occur in both the protein structure and the DNA molecule, making it essential to quantify and characterise these changes. This work aims to quantify and characterise the conformational changes in the protein structures. This is achieved by calculating the root-mean-square deviation of the protein structure in the free state and DNA bound state. A database is curated which has a significant number of DNA-bound and unbound pairs available. For the unbound structure of the proteins, pre-processing is required to remove chains other than those found in its corresponding DNA-bound structure followed by superimposing it to the DNA-bound structures obtained from the Protein Data Bank (PDB) to calculate the RMSD. After quantifying the conformational changes using RMSD values, the proteins are categorised into six types based on their observed conformational changes. In this study, the primary objective is to conduct a comprehensive analysis of conformational changes in DNA-binding proteins and investigate their correlation with various protein properties. By exploring these correlations, the study aims to unravel potential patterns or dependencies between the extent of conformational changes and the diverse properties of DNA-binding proteins. Such insights can shed light on the functional implications of structural variations in DNA-protein interactions, providing valuable information about how protein dynamics contribute to DNA recognition and binding. In addition, this work endeavours to broaden its scope by attempting to replicate DNA-bound complexes through the utilisation of freely available structures. By doing so, it aims to assess the reliability and accuracy of docking methods in predicting and reconstructing these complexes. The primary objective is to evaluate the feasibility and effectiveness of such techniques in the context of complex formation between DNA and protein molecules.

Distinctive Features of the XBB.1.5 and XBB.1.16 Spike Protein Receptor-Binding Domains and Their Roles in Conformational Changes and Angiotensin-Converting Enzyme 2 Binding.

The emergence and the high transmissibility of the XBB.1.5 and XBB.1.16 subvariants of the SARS-CoV-2 omicron has reignited concerns over the potential impact on vaccine efficacy for these and future variants. We investigated the roles of the XBB.1.5 and XBB.1.16 mutations on the structure of the spike protein's receptor-binding domain (RBD) and its interactions with the host cell receptor ACE2. To bind to ACE2, the RBD must transition from the closed-form to the open-form configuration. We found that the XBB variants have less stable closed-form structures that may make the transition to the open-form easier. We found that the mutations enhance the RBD-ACE2 interactions in XBB.1.16 compared to XBB.1.5. We observed significant structural changes in the loop and motif regions of the RBD, altering well-known antibody-binding sites and potentially rendering primary RBD-specific antibodies ineffective. Our findings elucidate how subtle structural changes and interactions contribute to the subvariants' fitness over their predecessors.

N-Salicyl-AAn-picolamide Foldameric Peptides Exhibit Quorum Sensing Inhibition of Pseudomonas aeruginosa (PA14).

An organic acid, salicylic acid, and its derivatives are constituents of various natural products possessing remarkable bioactivity. O-Acetyl salicylate (aspirin) is a well-known life-saving drug. Its peptide derivative salicylamide has also been explored in the designing of peptide-based therapeutic drugs. An organic base, picolylamine has been recently explored for designing diagnostic probes. However, both the acid and base have common features as metal chelating with coordinating metals. Thus, these scaffolds could be used for designing inhibitors of various metalloenzymes. Their characteristic properties encourage us to design peptides containing both scaffolds (salicylic acid and picolylamine) at opposite terminals. So far there is no report available on such conjugated peptides. This report describes the synthesis, conformational analysis, and biochemical assessment of rationally designed N-salicyl-AAn-picolamide peptides. Pleasantly, we have obtained the crystal structures of representative peptides that confirm their roles in conformational changes. Our biological assessment as quorum sensing inhibitors has revealed that their di/tripeptides inhibit quorum sensing of the pathogenic bacterium PA14 strain. Hence, these peptides have promising foldameric and therapeutic values.

The role of conformational change and key glutamic acid residues in the ClC-ec1 antiporter

The triple glutamine (Q) mutant (QQQ) structure of a Cl−/H+ antiporter from Escherichia coli (ClC-ec1) displaying a novel backbone arrangement has been used to challenge the long-held notion that Cl−/H+ antiporters do not operate through large conformational motions. The QQQ mutant substitutes the glutamine residue for an external glutamate E148, an internal glutamate E203, and a third glutamate E113 that hydrogen-bonds with E203. However, it is unknown if QQQ represents a physiologically relevant state, as well as how the protonation of the wild-type glutamates relates to the global dynamics. We herein apply continuous constant-pH molecular dynamics to investigate the H+-coupled dynamics of ClC-ec1. Although any large-scale conformational rearrangement upon acidification would be due to the accumulation of excess charge within the protein, protonation of the glutamates significantly impacts mainly the local structure and dynamics. Despite the fact that the extracellular pore enlarges at acidic pHs, an occluded ClC-ec1 within the active pH range of 3.5–7.5 requires a protonated E148 to facilitate extracellular Cl− release. E203 is also involved in the intracellular H+ transfer as an H+ acceptor. The water wire connection of E148 with the intracellular solution is regulated by the charge states of the E113/E203 dyad with coupled proton titration. However, the dynamics extracted from our simulations are not QQQ-like, indicating that the QQQ mutant does not represent the behavior of the wild-type ClC-ec1. These findings reinforce the necessity of having a protonatable residue at the E203 position in ClC-ec1 and suggest that a higher level of complexity exists for the intracellular H+ transfer in Cl−/H+ antiporters.

Molecular plasticity of CBM3 ancillary domain leads to conformational changes in the cellulose binding interface

Carbohydrate-binding modules (CBMs) constitute independently folded domains typically associated with carbohydrate-active enzymes (CAZymes). These modules are considered to have a rigid structure without notable conformational changes upon ligand binding, exhibiting a complementary topography in relation to the target carbohydrate. Herein, the high-resolution SAD-solved structure of a CBM from family 3 (BsCBM3) that binds to crystalline cellulose is reported in two crystalline forms. This module showed molecular plasticity with structural differences detected between the two crystalline forms and high RMSD values when compared to NMR ensemble of models. Pronounced structural variances were observed in the cellulose binding interface between NMR and XTAL structures, which were corroborated by molecular dynamics simulations. These findings support that family 3 CBMs targeting to cellulose are rather structurally dynamic modules than rigid entities, suggesting a potential role of conformational changes in polysaccharide recognition and modulation of enzyme activity.

Design and interpretation of experiments to establish enzyme pathway and define the role of conformational changes in enzyme specificity.

We describe the experimental methods and analysis to define the role of enzyme conformational changes in specificity based on published studies using DNA polymerases as an ideal model system. Rather than give details of how to perform transient-state and single-turnover kinetic experiments, we focus on the rationale of the experimental design and interpretation. We show how initial experiments to measure kcat and kcat/Km can accurately quantify specificity but do not define its underlying mechanistic basis. We describe methods to fluorescently label enzymes to monitor conformational changes and to correlate fluorescence signals with rapid-chemical-quench flow assays to define the steps in the pathway. Measurements of the rate of product release and of the kinetics of the reverse reaction complete the kinetic and thermodynamic description of the full reaction pathway. This analysis showed that the substrate-induced change in enzyme structure from an open to a closed state was much faster than rate-limiting chemical bond formation. However, because the reverse of the conformational change was much slower than chemistry, specificity is governed solely by the product of the binding constant for the initial weak substrate binding and the rate constant for the conformational change (kcat/Km=K1k2) so that the specificity constant does not include kcat. The enzyme conformational change leads to a closed complex in which the substrate is bound tightly and is committed to the forward reaction. In contrast, an incorrect substrate is bound weakly, and the rate of chemistry is slow, so the mismatch is released from the enzyme rapidly. Thus, the substrate-induced-fit is the major determinant of specificity. The methods outlined here should be applicable to other enzyme systems.

Abstract 2401: Applying the Brakes: Understanding the Role of Conformational Changes in Kinesin-5

Abstract 2401: Applying the Brakes: Understanding the Role of Conformational Changes in Kinesin-5

Determination of rate constants for conformational changes of RNA helicases by single-molecule FRET TIRF microscopy.

RNA helicases couple nucleotide-driven conformational changes to the unwinding of RNA duplexes. Interaction partners can regulate helicase activity by altering the rate constants of these conformational changes. Single-molecule FRET experiments on donor/acceptor-labeled, immobilized molecules are ideally suited to monitor conformational changes in real time and to extract rate constants for these processes. This article provides guidance on how to design, perform, and analyze single-molecule FRET experiments by TIRF microscopy. It covers the theoretical background of FRET and single-molecule TIRF microscopy, the considerations to prepare proteins of interest for donor/acceptor labeling and surface immobilization, and the principles and procedures of data analysis, including image analysis and the determination of FRET time traces, the extraction of rate constants from FRET time traces, and the general conclusions that can be drawn from these data. A case study, using the DEAD-box protein eIF4A as an example, highlights how single-molecule FRET studies have been instrumental in understanding the role of conformational changes for duplex unwinding and for the regulation of helicase activities. Selected examples illustrate which conclusions can be drawn from the kinetic data obtained, highlight possible pitfalls in data analysis and interpretation, and outline how kinetic models can be related to functionally relevant states.

Polymer-solvent interaction and conformational changes at a molecular level: Implication to solvent-assisted deformation and aggregation at the polymer surface

HypothesisWe hypothesize that varying the chemical structure of the monomeric unit in a polymer will affect the surface structure and interfacial molecular group orientations of the polymer film leveraging its response to solvents of different chemical affinities. ExperimentsPoly (2-methoxy ethyl methacrylate) and poly (2-tertbutoxy ethyl methacrylate) thin films exposed to either deuterated water (D2O) or deuterated chloroform (CDCl3) were studied by sum frequency generation (SFG) spectroscopy, contact angle goniometry, and atomic force microscopy (AFM) at the polymer-solvent interface, supported with molecular simulation studies. FindingsSFG spectral analysis of the polymer thin films corroborated molecular re-organization at the surface when exposed to different chemical environments. The AFM height images of the polymer surfaces were homogeneously flat under CDCl3 and showed swollen regions under D2O. Following the removal of D2O, the exposed areas have imprinted, recessed locations and exposure to CDCl3 resulted in the formation of aggregates. The chemical affinity and characteristics of the solvents played a role in conformational change at the polymer surface. It had direct implications to interfacial processes involving adsorption, permeation which eventually leads to swelling, deformation or aggregation, and possibly dissolution.

Single-Molecule Sampling of Dihydrofolate Reductase Shows Kinetic Pauses and an Endosteric Effect Linked to Catalysis.

The ability to sample multiple reactions on the same single enzyme is important to link rare intermediates with catalysis and to unravel the role of conformational changes. Despite decades of efforts, however, the single-molecule characterization of nonfluorogenic enzymes during multiple catalytic turnovers has been elusive. Here, we show that nanopore currents allow sampling the dynamic exchange between five structural intermediates during E. coli dihydrofolate reductase (DHFR) catalysis. We found that an endosteric effect promotes the binding of the substrate to the enzyme with a specific hierarchy. The chemical step then switched the enzyme from the closed to the occluded conformation, which in turn promotes the release of the reduced cofactor NADP+. Unexpectedly, only a few reactive complexes lead to catalysis. Furthermore, second-long catalytic pauses were observed, possibly reflecting an off-path conformation generated during the reaction. Finally, the free energy from multiple cofactor binding events were required to release the product and switch DHFR back to the reactive conformer. This catalytic fueled concerted mechanism is likely to have evolved to improve the catalytic efficiency of DHFR under the high concentrations of NADP+ in E. coli and might be a general feature for complex enzymatic reactions where the binding and release of the products must be tightly controlled.

The eptinezumab:CGRP complex structure – the role of conformational changes in binding stabilization

ABSTRACT To further elucidate the mechanism of action and binding properties of eptinezumab to calcitonin gene-related peptide (CGRP), X-ray crystallography, computational alanine scanning, and molecular dynamics were used. X-ray diffraction data were collected to determine the three-dimensional structures of the unbound eptinezumab antigen-binding fragment (Fab) and the Fab:CGRP complex. Molecular dynamics simulations were performed to analyze the transition between uncomplexed and complex states. The amidated C-terminus of CGRP was shown to bind in a pocket formed by the Fab heavy and light chains. There was extensive contact between all six complementarity-determining regions (CDRs; composed of light-chain [L1, L2, and L3] and heavy-chain [H1, H2, H3]) of eptinezumab and CGRP. The complex demonstrated a high ligand-binding surface area dominated by aromatic residues. CDR L3 contains a disulfide bond that stabilizes the loop, contributes surface area to the binding pocket, and provides van der Waals contacts. Comparison of the uncomplexed and complex structures revealed motion near the binding cleft. The CDR loops H2 and H3 were displaced ~1.4–2.0 Å and residue H-Tyr33 changed conformation, creating a ‘latch-and-lock’ mechanism for binding CGRP and preventing dissociation. Computational alanine scanning of CGRP identified energetic ‘hot spots’ that contribute to binding energy; mutating these positions to residues in homologous neuropeptides resulted in unfavorable binding energies. The attributes of the Fab region and the conformational changes that occur in eptinezumab during binding to CGRP contribute to the specificity, durability, and strength of the interaction, and likely underlie the rapid and sustained migraine preventive effect observed in clinical studies.

Role of Conformational Changes of Hexameric Bacterial Uridine Phosphorylases in Substrate Binding

Role of Conformational Changes of Hexameric Bacterial Uridine Phosphorylases in Substrate Binding

Role of Conformational Change and Glucose Binding Sites in the Enhanced Glucose Tolerance of Agrobacterium tumefaciens 5A GH1 β-Glucosidase Mutants.

β-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent Ki,Glc = 686 mM). We report our steady-state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose-protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.

Molecular level insights into the inhibition of gene expression by thiamine pyrophosphate (TPP) analogs for TPP riboswitch: A well-tempered metadynamics simulations study

Riboswitches are metabolite sensing aptamer domains present in non-coding regions in RNA and act as gene-regulating elements. Thiamine pyrophosphate (TPP) riboswitch is evolved as a new target for developing antibiotics against many pathogenic bacteria. The earlier reports suggest that the modification of the pyrophosphate group in the ligand molecule can enhance gene expression. In this work, we have examined the binding affinity and efficacy of TPP and two recently reported ligands, CH2-TPP, and CF2-TPP, using Well-tempered metadynamics (WT-MtD) simulations. The experimental in vitro assays show that both TPP and CH2-TPP repress the gene expression to the same extent. The calculated binding energies correlate well with the experimental study and show the same trend of binding affinity of ligands for the TPP riboswitch. The root mean square fluctuation profiles suggest that the CH2-TPP and TPP trigger higher fluctuations in P1 and L3 region, and such fluctuations in the P1 region is involved in the gene regulation process. The metal ion mediated contact of TPP ligand with pyrophosphate binding helix is found to be critical in the gene regulation process. The simulation results corroborate the experimental observations that the role of conformational changes occurring in different riboswitch regions upon ligand binding is essential to repress the gene expression process. This work sheds light on the subtle change in the ligand structure that can induce a more considerable impact on binding affinity and efficacy of ligands with riboswitch.

Structural and molecular dynamic studies of Pseudomonas aeruginosa OdaA reveal the regulation role of a C-terminal hinge element

BackgroundCrotonase superfamily members exhibit great catalytic diversity towards various acyl-CoA substrates. A common CoA moiety binding pattern is usually observed in this family, understanding the substrate-binding mechanism would facilitate the rational engineering of crotonases for improved properties. MethodsWe applied X-ray crystallography to investigate a putative enoyl-CoA hydratase/isomerase OdaA in Pseudomonas aeruginosa. Thermal shift assay (TSA) were performed to explore the binding of OdaA with CoA thioester substrates. Furthermore, we performed molecular dynamics (MD) simulations to elucidate the dynamics of its CoA-binding site. ResultsWe solved the crystal structures of the apo and CoA-bound OdaA. Thermal shift assay (TSA) showed that CoA thioester substrates bind to OdaA with a different degree. MD simulations demonstrated that the C-terminal alpha helix underwent a structural transition and a hinge region would associate with this conformational change. ConclusionsTSA in combination with MD simulations elucidate that the dynamics of C-terminal alpha helix in CoA-binding, and a hinge region play an important role in conformational change. General significanceThose results help to extend our knowledge about the nature of crotonases and would be informative for future mechanistic studies and industry applications.

The role of ligand-gated conformational changes in enzyme catalysis.

Structural and biochemical studies on diverse enzymes have highlighted the importance of ligand-gated conformational changes in enzyme catalysis, where the intrinsic binding energy of the common phosphoryl group of their substrates is used to drive energetically unfavorable conformational changes in catalytic loops, from inactive open to catalytically competent closed conformations. However, computational studies have historically been unable to capture the activating role of these conformational changes. Here, we discuss recent experimental and computational studies, which can remarkably pinpoint the role of ligand-gated conformational changes in enzyme catalysis, even when not modeling the loop dynamics explicitly. Finally, through our joint analyses of these data, we demonstrate how the synergy between theory and experiment is crucial for furthering our understanding of enzyme catalysis.

A Central Region of NF-κB Essential Modulator Is Required for IKKβ-Induced Conformational Change and for Signal Propagation.

NF-κB essential modulator (NEMO) regulates NF-κB signaling by acting as a scaffold for the kinase IKKβ to direct its activity toward the NF-κB inhibitor, IκBα. Here, we show that a highly conserved central region of NEMO termed the intervening domain (IVD, amino acids 112-195) plays a key role in NEMO function. We determined a structural model of full-length NEMO by small-angle X-ray scattering and show that full-length, wild-type NEMO becomes more compact upon binding of a peptide comprising the NEMO binding domain of IKKβ (amino acids 701-745). Mutation of conserved IVD residues (9SG-NEMO) disrupts this conformational change in NEMO and abolishes the ability of NEMO to propagate NF-κB signaling in cells, although the affinity of 9SG-NEMO for IKKβ compared to that of the wild type is unchanged. On the basis of these results, we propose a model in which the IVD is required for a conformational change in NEMO that is necessary for its ability to direct phosphorylation of IκBα by IKKβ. Our findings suggest a molecular explanation for certain disease-associated mutations within the IVD and provide insight into the role of conformational change in signaling scaffold proteins.