Internal Conformational Changes Research Articles

Deciphering the Impact of Mutations on the Binding Efficacy of SARS-CoV-2 Omicron and Delta Variants With Human ACE2 Receptor.

The pandemic of COVID-19, caused by SARS-CoV-2, has globally affected the human health and economy. Since the emergence of the novel coronavirus SARS-CoV-2, the life-threatening virus continues to mutate and evolve. Irrespective of acquired natural immunity and vaccine-induced immunity, the emerging multiple variants are growing exponentially, crossing the territorial barriers of the modern world. The rapid emergence of SARS-CoV-2 multiple variants challenges global researchers regarding the efficacy of available vaccines and variant transmissibility. SARS-CoV-2 surface-anchored S-protein recognizes and interacts with the host-cell ACE2, facilitating viral adherence and entrance into the cell. Understanding the interfacial interactions between the spike protein of SARS-CoV-2 variants and human ACE2 receptor is important for the design and development of antiviral therapeutics against SARS-CoV-2 emerging variants. Despite extensive research, the crucial determinants related to the molecular interactions between the spike protein of SARS-CoV-2 variants and host receptors are poorly understood. Thus, in this study, we explore the comparative interfacial binding pattern of SARS-CoV-2 spike RBD of wild type, Delta, and Omicron with the human ACE2 receptor to determine the crucial determinants at the atomistic level, using MD simulation and MM/GBSA energy calculations. Based on our findings, the substitution of Q493R, G496S, Q498R, and Y505H induced internal conformational changes in Omicron spike RBD, which leads to higher binding affinity than Delta spike RBD with the human ACE2 receptor, eventually contributing to higher transmission and infectivity. Taken together, these results could be used for the structure-based design of effective antiviral therapeutics against SARS-CoV-2 variants.

Cotemporal Single-Molecule Force and Fluorescence Measurements to Determine the Mechanism of Ribosome Translocation.

Ribosomes are at the core of the central dogma of life. They perform the last major step of gene expression by translating the information written in the nucleotide codon sequences into the amino acid sequence of a protein. This is a complex mechanochemical process that requires the coordination of multiple dynamic events within the ribosome such as the precise timing of decoding and the subsequent translocation along the mRNA. We have previously used a high-resolution optical tweezers instrument with single-molecule fluorescence capabilities ("fleezers") to study how ribosomes couple binding of the GTPase translation elongation factor EF-G with internal conformational changes to unwind and progress across the mechanical barriers posed by mRNA secondary structures. Here, we present a detailed description of the procedures for monitoring two orthogonal channels (EF-G binding and translocation) by single actively translating ribosomes in real-time, to uncover the mechanism by which they harness chemical energy to generate mechanical force and displacement.

Molecular mechanism concerning conformational changes of CDK2 mediated by binding of inhibitors using molecular dynamics simulations and principal component analysis

ABSTRACT Cyclin-dependent kinase 2 (CDK2) has been regarded as a promising drug target for anti-tumour agents. In this study, molecular dynamics (MD) simulations and principal component (PC) analysis were used to explore binding mechanism of three inhibitors 1PU, CDK, 50Z to CDK2 and influences of their bindings on conformational changes of CDK2. The results show that bindings of inhibitors yield obvious impacts on internal dynamics, movement patterns and conformational changes of CDK2. In addition, molecular mechanics generalized Born surface area (MM-GBSA) was applied to calculate binding free energies between three inhibitors and CDK2 and evaluate their binding ability to CDK2. The results show that CDK has the strongest binding to CDK2 among the current three inhibitors. Residue-based free energy decomposition method was further utilized to decode the contributions of a single residue to binding of inhibitors, and it was found that three inhibitors not only produce hydrogen bonding interactions and hydrophobic interactions with key residues of CDK2, which promotes binding of three inhibitors to CDK2, but also share similar binding modes. This work is expected to be helpful for design of efficient drugs targeting CDK2.

Frontier Expansion Sampling: A Method to Accelerate Conformational Search by Identifying Novel Seed Structures for Restart.

Traditional molecular dynamics (MD) simulations have difficulties in tracking the slow molecular motions, at least partially due to the waste of sampling in already sampled regions. Here, we proposed a new enhanced sampling method, frontier expansion sampling (FEXS), to improve the sampling efficiency of molecular simulations by iteratively selecting seed structures diversely distributed at the "frontier" of an already sampled region to initiate new simulations. Different from other enhanced sampling methods, FEXS identifies the "frontier" seeds by integrating the Gaussian mixture model and the convex hull algorithm, which effectively improves the structural variation among the selected seeds and thus the descendant simulations. Validation in three protein systems, including the folding of chignolin, open-to-closed transition of maltodextrin binding protein, and internal conformational change of bovine pancreatic trypsin inhibitor, confirmed the effectiveness of this novel method in enhancing the sampling of conventional MD simulations to observe the large-scale protein conformational changes. When compared with other enhanced sampling methods like the structural dissimilarity sampling (SDS), FEXS reached at least the same level of sampling efficiency but was capable of providing complementary information in the three tested protein systems.

Molecular simulation of folding and aggregation of multi-core polycyclic aromatic compounds

Compared with polycyclic aromatic compounds (PACs) that have a single polyaromatic core, those having multiple cores connected by aliphatic chains can undergo significant internal conformational change, leading to folding, the stacking between the cores within the same molecule. In this work, we investigated this interesting phenomenon using molecular dynamics simulations, performed in three different solvents: water, heptane and toluene. In addition, by having several multi-core PACs in the same solution, the relationship between intramolecular folding and intermolecular aggregation was elucidated. In water, a single multi-core PAC preferred to stay in the unfolded state; contrarily, folded configuration became dominant when multiple molecules aggregated. In heptane and toluene, a multi-core PAC molecule could switch easily between folded and unfolded configurations, regardless of whether it was in single dispersed or aggregated states. Introduction of single-core PACs further modified the folding characteristics of multi-core PACs in water and heptane. This work provided fundamental insights into the interplay between folding, homoaggregation (between multi-core PACs) and heteroaggregation (between multi-core and single-core PACs) in a colloidal system.

Co-temporal Force and Fluorescence Measurements Reveal a Ribosomal Gear Shift Mechanism of Translation Regulation by Structured mRNAs.

The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.

Differential cellular contractility as a mechanism for stiffness sensing

The ability of cells to sense and respond to the mechanical properties of their environments is fundamental to a range of cellular behaviours, with substrate stiffness increasingly being found to be a key signalling factor. Although active contractility of the cytoskeleton is clearly necessary for stiffness sensing in cells, the physical mechanisms connecting contractility with mechanosensing and molecular conformational change are not well understood. Here we present a contractility-driven mechanism for linking changes in substrate stiffness with internal conformational changes. Cellular contractility is often assumed to imply an associated compressive strain. We show, however, that where the contractility is non-uniform, localized areas of internal stretch can be generated as stiffer substrates are encountered. This suggests a physical mechanism for the stretch-activation of mechanotransductive molecules on stiffer substrates. Importantly, the areas of internal stretch occur deep within the cell and not near the cellular perimeter, which region is more traditionally associated with stiffness sensing through e.g. focal adhesions. This supports recent experimental results on whole-cell mechanically-driven mechanotransduction. Considering cellular shape we show that aspect ratio acts as an additional control parameter, so that the onset of positive strain moves to higher stiffness values in elliptical cells.

Elastin-like Peptide in Confinement: FT-IR and NMR T 1 Relaxation Data

Abstract We employed FT-IR and NMR experiments to investigate the influence of a cell-mimicking crowding environment on the structure and dynamics of an elastin-like peptide (ELP) with the sequence GVG(VPGVG)3, which – due to a high number of hydrophobic amino acid side chains – exhibits an inverse temperature transition (ITT). As simplified crowding agent, we used 30 wt% Ficoll. The FT-IR data revealed the well-known broad ITT above ~25°C, as observed by the decrease of the relative population of random coil structures and the concomitant increase of type II β-turns. Interestingly, the addition of Ficoll leads to a destabilizing effect of type II β-turn structures. This is in contrast to the expected excluded-volume effect of the macromolecular crowder, but can be explained by weak interactions of the peptide with the polysaccharide chains of the crowding agent. Further, the crowding agent leads to the onset of a reversal of the folding transition at high temperatures. The full assignment of the ELP allowed for a residue-specific investigation of the dynamic behavior of ELP by NMR. Due to a strong change of microscopic viscosity between native/buffered conditions and crowded conditions, relaxation data remain inconclusive with respect to the observation of an ITT. Hence, no quantitative details in terms of internal conformational changes can be obtained. However, temperature dependent differences in the 13C relaxation behavior between core and terminal parts of the peptide indicate temperature induced changes in the internal dynamics with generally higher internal mobility at chain ends: This is in full agreement with FT-IR data. In harmony with the FT-IR analysis, macromolecular crowding does not lead to significant changes in the relaxation behavior.

Observing Single-Molecule Dynamics at Millimolar Concentrations.

Single-molecule fluorescence microscopy is a powerful tool for revealing chemical dynamics and molecular association mechanisms, but has been limited to low concentrations of fluorescent species and is only suitable for studying high affinity reactions. Here, we combine nanophotonic zero-mode waveguides (ZMWs) with fluorescence resonance energy transfer (FRET) to resolve single-molecule association dynamics at up to millimolar concentrations of fluorescent species. This approach extends the resolution of molecular dynamics to >100-fold higher concentrations, enabling observations at concentrations relevant to biological and chemical processes, and thus making single-molecule techniques applicable to a tremendous range of previously inaccessible molecular targets. We deploy this approach to show that the binding of cGMP to pacemaking ion channels is weakened by a slower internal conformational change.

Observing Single‐Molecule Dynamics at Millimolar Concentrations

AbstractSingle‐molecule fluorescence microscopy is a powerful tool for revealing chemical dynamics and molecular association mechanisms, but has been limited to low concentrations of fluorescent species and is only suitable for studying high affinity reactions. Here, we combine nanophotonic zero‐mode waveguides (ZMWs) with fluorescence resonance energy transfer (FRET) to resolve single‐molecule association dynamics at up to millimolar concentrations of fluorescent species. This approach extends the resolution of molecular dynamics to >100‐fold higher concentrations, enabling observations at concentrations relevant to biological and chemical processes, and thus making single‐molecule techniques applicable to a tremendous range of previously inaccessible molecular targets. We deploy this approach to show that the binding of cGMP to pacemaking ion channels is weakened by a slower internal conformational change.

Single Molecule Fluorescence Studies on Nucleosome Dynamics

Nucleosomes, as the basic packing unit of chromatin, regulate DNA accessibility and have significant influence on gene expression. Two copies of each histone protein (H2A, H2B, H3, H4) build up the protein octamer, around which approximately 150 bp of DNA are wrapped.The N-terminal tails of the histones protrude from the nucleosome; they are important for inter- and intranucleosomal interactions. Nucleosome disassembly may be forced in vitro by increasing salt concentration and may be followed by Forster resonance energy transfer (FRET) between fluorophores. We use reconstituted Xenopus laevis nucleosomes, fluorescently labeled on different positions[2] to compare the disassembly of nucleosomes containing mutated histones or modifications to the wild type.Here we focus on the dynamics of the N-tail of H3. Mutated versions of H3 labeled with Alexa488 were used to examine whether there are interactions between the labeled H3 tail and the labeled DNA or other labeled histones. First results show an interaction of the H3 tail and the DNA in vicinity of the dyad axis.The second part is about the functional relevance of a particular region of H2A. Molecular dynamic simulations on ‘tailless’ variants of H3 and H2A have suggested conformational changes affecting two arginines of H2A. Furthermore a conformational change of the protruding part of the DNA was described, which is due to internal conformational changes of H2A[3].From those observations the question arises whether these amino acids are necessary for stable octamers and/or nucleosomes, and how they influence DNA breathing, unwrapping, and stability of entire nucleosomes. To address these questions we generated recombinant H2A proteins incorporating site-specific mutations (R81A, R88A, R81AR88A, R81E, R88E, R81ER88E). Our results show a decreasing stability associated with position (Wt > R88 > R81 > R81R88) and charge (RA > RE) of the amino acids.

Monte Carlo simulations of peptide-membrane interactions with the MCPep web server

The MCPep server (http://bental.tau.ac.il/MCPep/) is designed for non-experts wishing to perform Monte Carlo (MC) simulations of helical peptides in association with lipid membranes. MCPep is a web implementation of a previously developed MC simulation model. The model has been tested on a variety of peptides and protein fragments. The simulations successfully reproduced available empirical data and provided new molecular insights, such as the preferred locations of peptides in the membrane and the contribution of individual amino acids to membrane association. MCPep simulates the peptide in the aqueous phase and membrane environments, both described implicitly. In the former, the peptide is subjected solely to internal conformational changes, and in the latter, each MC cycle includes additional external rigid body rotational and translational motions to allow the peptide to change its location in the membrane. The server can explore the interaction of helical peptides of any amino-acid composition with membranes of various lipid compositions. Given the peptide’s sequence or structure and the natural width and surface charge of the membrane, MCPep reports the main determinants of peptide–membrane interactions, e.g. average location and orientation in the membrane, free energy of membrane association and the peptide’s helical content. Snapshots of example simulations are also provided.

Von Willebrand factor folds into a bouquet

The large glycoprotein von Willebrand factor (vWF) serves an important role in orchestrating the blood vessel9s response to injury. It is released from activated endothelial cells and forms long multimeric strings that bind exposed extracellular matrix and circulating platelets and thereby initiate the formation of a platelet plug sealing the wound. Central to this process is the rapid transition of multimeric vWF from a tightly packed storage form present in acidic intraendothelial granules, to the elongated string that is active at neutral vascular pH. In this issue of The EMBO Journal , Zhou and coworkers now provide compelling structural evidence for internal conformational changes that accompany this transition and thereby serve important regulatory functions. They show that the C‐terminal part of the protein zips up into a dimeric bouquet at the acidic internal pH of the storage granules but opens up at the neutral pH of plasma, thereby assisting the establishment of elongated vWF strings capable of efficiently capturing platelets.

Interplay of mechanical and binding properties of Fibronectin type I.

Fibronectins (FNs) are a major component of the extracellular matrix (ECM), and provide important binding sites for a variety of ligands outside and on the surface of the cell. Similar to other ECM proteins, FNs are consistently subject to mechanical stress in the ECM. Therefore, it is important to study their structure and binding properties under mechanical stress and understand how their binding and mechanical properties might affect each other. Although certain FN modules have been extensively investigated, no simulation studies have been reported for the FN type I (Fn1) domains, despite their prominent role in binding of various protein modules to FN polymers in the ECM. Using equilibrium and steered molecular dynamics simulations, we have studied mechanical properties of Fn1 modules in the presence or the absence of a specific FN-binding peptide (FnBP). We have also investigated how the binding of the FnBP peptide to Fn1 might be affected by tensile force. Despite the presence of disulfide bonds within individual Fn1 modules that are presumed to prevent their extension, it is found that significant internal structural changes within individual modules are induced by the forces applied in our simulations. These internal structural changes result in significant variations in the accessibility of different residues of the Fn1 modules, which affect their exposure, and, thus, the binding properties of the Fn1 modules. Binding of the FnBP appears to reduce the flexibility of the linker region connecting individual Fn1 modules (exhibited in the form of reduced fluctuation and motion of the linker region), both with regard to bending and stretching motions, and hence stabilizes the inter-domain configuration under force. Under large tensile forces, the FnBP peptide unbinds from Fn1. The results suggest that Fn1 modules in FN polymers do contribute to the overall extension caused by force-induced stretching of the polymer in the ECM, and that binding properties of Fn1 modules can be affected by mechanically induced internal protein conformational changes in spite of the presence of disulfide bonds which were presumed to completely abolish the capacity of Fn1 modules to undergo extension in response to external forces.

First passage time distribution for the 1D diffusion of particles with internal degrees of freedom

Proteins searching for their targets on DNA do so by non-specifically adsorbing and sliding along DNA. It became known recently that the proteins simultaneously undergo some internal conformational changes (partial folding and unfolding), such that in some of the sub-states their ability to recognize the target site is enhanced at the expense of diffusivity which is suppressed. We analyze the probability distribution of the first passage times of these proteins into the target taking into account the internal sub-states of proteins. We show that there are two distinct scaling regimes: one is diffusion controlled and the other is absorption controlled.

EPAC proteins transduce diverse cellular actions of cAMP

It has now been over 10 years since efforts to completely understand the signalling actions of cAMP (3'-5'-cyclic adenosine monophosphate) led to the discovery of exchange protein directly activated by cAMP (EPAC) proteins. In the current review we will highlight important advances in the understanding of EPAC structure and function and demonstrate that EPAC proteins mediate multiple actions of cAMP in cells, revealing future targets for pharmaceutical intervention. It has been known for some time that drugs that elevate intracellular cAMP levels have proven therapeutic benefit for diseases ranging from depression to inflammation. The challenge now is to determine which of these positive actions of cAMP involve activation of EPAC-regulated signal transduction pathways. EPACs are specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, which they activate independently of the classical routes for cAMP signalling, cyclic nucleotide-gated ion channels and protein kinase A. Rather, EPAC activation is triggered by internal conformational changes induced by direct interaction with cAMP. Leading from this has been the development of EPAC-specific agonists, which has helped to delineate numerous cellular actions of cAMP that rely on subsequent activation of EPAC. These include regulation of exocytosis and the control of cell adhesion, growth, division and differentiation. Recent work also implicates EPAC in the regulation of anti-inflammatory signalling in the vascular endothelium, namely negative regulation of pro-inflammatory cytokine signalling and positive support of barrier function. Further elucidation of these important signalling mechanisms will no doubt support the development of the next generation of anti-inflammatory drugs.

The structure of the Au(111)/methylthiolate interface: New insights from near-edge x-ray absorption spectroscopy and x-ray standing waves

The local structure of the Au(111)(square root(3) x square root(3))R30 degrees-methylthiolate surface phase has been investigated by S K-edge near-edge s-ray absorption fine structure (NEXAFS) both experimentally and theoretically and by experimental normal-incidence x-ray standing waves (NIXSW) at both the C and S atomic sites. NEXAFS shows not only excitation into the intramolecular sigma(*) S-C resonance but also into a sigma(*) S-Au orbital perpendicular to the surface, clearly identifying the local S headgroup site as atop a Au atom. Simulations show that it is not possible, however, to distinguish between the two possible adatom reconstruction models; a single thiolate species atop a hollow-site Au adatom or a dithiolate moiety comprising two thiolate species bonded to a bridge-bonded Au adatom. Within this dithiolate moiety a second sigma(*) S-Au orbital that lies near parallel to the surface has a higher energy that overlaps that of the sigma(*) S-C resonance. The new NIXSW data show the S-C bond to be tilted by 61 degrees relative to the surface normal, with a preferred azimuthal orientation in <211>, corresponding to the intermolecular nearest-neighbor directions. This azimuthal orientation is consistent with the thiolate being atop a hollow-site Au adatom, but not consistent with the originally proposed Au-adatom-dithiolate moiety. However, internal conformational changes within this species could, perhaps, render this model also consistent with the experimental data.

A novel method for dissolution and stabilization of non‐mulberry silk gland protein fibroin using anionic surfactant sodium dodecyl sulfate

The importance of silk protein has increased because of its potential use as a natural biopolymer for tissue engineering and biomedical applications. In this report we show a novel and ecofriendly method for dissolution of gland silk protein fibroin. Non-mulberry silk fibroin from mature fifth instar larvae of Antheraea mylitta was found to be optimally soluble in 1% (w/v) anionic surfactant sodium dodecyl sulfate (SDS). Regenerated fibroin showed distinct bands of approximately 395 and 197 kDa on electrophoresis in non-reducing and reducing conditions, respectively. Enhanced fibroin dissolution via internalization of hydrophobic amino groups inside a hydrophilic amino acid core in the form of micelles was observed. Prolonged storage stability without gelation of SDS-extracted fibroin was seen. Atomic force microscopy showed micellar aggregation with mean micellar aggregation size of 8 nm. Circular dichroism spectroscopy revealed predominantly helical conformation due to surfactant addition with internal protein conformational changes as revealed by fluorescence spectroscopic studies.

Rouse Model with Internal Friction: A Coarse Grained Framework for Single Biopolymer Dynamics

Recent work has highlighted an intimate link between the internal friction of single biopolymers and dynamics on an underlying energy landscape of conformational change. Here, we examine the coarse-grained dynamics of flexible biopolymers by incorporating local internal friction sources into the successful Rouse model of polymer dynamics. Our main result is a closed-form expression of the frequency response function of a Rouse chain with and without internal friction. We find that the regimes in which internal friction and solvent friction dominate are characterized by single-mode dumbbell (|J(ω → ∞)| ∼ ω-1) and Rouse-like behavior (|J(ω → ∞)| ∼ ω-1/2), respectively. A real-space analysis shows these behaviors are due to a frequency-dependent length of chain that can respond to perturbations. By distinguishing dissipation due to internal conformational change with that from the solvent, the RIF model provides a simple and generic basis for probing the structure and dynamics of single biomolecules in stretching experiments.

Two lutein molecules in LHCII have different conformations and functions: Insights into the molecular mechanism of thermal dissipation in plants

When LHCII forms aggregates, the internal conformational changes will result in chlorophyll fluorescence quenching. Uncovering the molecular mechanism of this phenomenon will help us to understand how plants dissipate the excess excitation energy through non-photochemical quenching (NPQ) process. The crystal structure of spinach and pea LHCII have been published, and recently, we solved another crystal structure of LHCII from cucumber at 2.66 Å resolution. Here we present the first direct structural evidence indicating that the two lutein(Lut) molecules bound in each LHCII monomer have different conformations, Lut621 has a more twisted conformation than that of Lut620. The intimate interaction between the Lut620 and Chl a612/Chl a611 dimer leads to form a hetero-trimer, which is considered to be a potential quenching site. We also discovered that the dehydration of the LHCII crystals resulted in a notable shrinkage of the crystal unit cell dimensions which was accompanied by a red-shift of the fluorescence emission spectra of the crystals. These phenomena suggest the changes in the crystal packing during dehydration might be the cause of internal conformational changes within LHCII. We proposed a conformational change related NPQ model based on the structure analysis.