Protein Motions Research Articles

Protein-DNA target search relies on quantum walk

Protein-DNA interactions play a fundamental role in all life systems. A critical issue of such interactions is given by the strategy of protein search for specific targets on DNA. The mechanisms by which the protein are able to find relatively small cognate sequences, typically 15–20 base pairs (bps) for repressors, and 4–6 bps for restriction enzymes among the millions of bp of non-specific chromosomal DNA have hardly engaged researchers for decades. Recent experimental studies have generated new insights on the basic processes of protein-DNA interactions evidencing the underlying complex dynamic phenomena involved, which combine three-dimensional and one-dimensional motion along the DNA chain. It has been demonstrated that protein molecules have an extraordinary ability to find the target very quickly on the DNA chain, in some cases, with two orders of magnitude faster than the diffusion limit. This unique property of protein-DNA search mechanism is known as facilitated diffusion. Several theoretical mechanisms have been suggested to describe the origin of facilitated diffusion. However, none of such models currently has the ability to fully describe the protein search strategy.In this paper, we suggest that the ability of proteins to identify consensus sequences on DNA is based on the entanglement of π-π electrons between DNA nucleotides and protein amino acids. The π-π entanglement is based on Quantum Walk (QW), through Coin-position entanglement (CPE). First, the protein identifies a dimer belonging to the consensus sequence, and localize a π on such dimer, hence, the other π electron scans the DNA chain until the sequence is identified. Focusing on the example of recognition of consensus sequences of EcoRV or EcoRI, we will describe the quantum features of QW on protein-DNA complexes during the search strategy, such as walker quadratic spreading on a coherent superposition of different vertices and environment-supported long-time survival probability of the walker. We will employ both discrete- or continuous-time versions of QW. Biased and unbiased classical Random Walk (CRW) have been used for a long time to describe the Protein-DNA search strategy. QW, the quantum version of CRW, has been widely studied for its applications in quantum information applications. In our biological application, the walker (the protein) resides at a vertex in a graph (the DNA structural topology). Differently to CRW, where the walker moves randomly, the quantum walker can hop along the edges in the graph to reach other vertices entering coherently a superposition across different vertices spreading quadratically faster than CRW analogous evidencing the typical speed up features of the QW. When applied to a protein-DNA target search problem, QW gives the possibility to achieve the experimental diffusional motion of proteins over diffusion classical limits experienced along DNA chains exploiting quantum features such as CPE and long-time survival probability supported by the environment. In turn, we come to the conclusion that, under quantum picture, the protein search strategy does not distinguish between one-dimensional (1D) and three-dimensional (3D) cases.

Exploring Large Domain Motions in Proteins Using Atomistic Molecular Dynamics with Enhanced Conformational Sampling.

Conformational transitions in multidomain proteins are essential for biological functions. The Apo conformations are typically open and flexible, while the Holo states form more compact conformations stabilized by protein-ligand interactions. Unfortunately, the atomically detailed mechanisms for such open-closed conformational changes are difficult to be accessed experimentally as well as computationally. To simulate the transitions using atomistic molecular dynamics (MD) simulations, efficient conformational sampling algorithms are required. In this work, we propose a new approach based on generalized replica-exchange with solute tempering (gREST) for exploring the open-closed conformational changes in multidomain proteins. Wherein, selected surface charged residues in a target protein are defined as the solute region in gREST simulation and the solute temperatures are different in replicas and exchanged between them to enhance the domain motions. This approach is called gREST selected surface charged residues (gREST_SSCR) and is applied to the Apo and Holo states of ribose binding protein (RBP) in solution. The conformational spaces sampled with gREST_SSCR are much wider than those with the conventional MD, sampling open-closed conformational changes while maintaining RBP domains’ stability. The free-energy landscapes of RBP in the Apo and Holo states are drawn along with twist and hinge angles of the two moving domains. The inter-domain salt-bridges that are not observed in the experimental structures are also important in the intermediate states during the conformational changes.

Conformational Plasticity in Human Heme-Based Dioxygenases.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and human tryptophan dioxygenase (hTDO) are two important heme proteins that degrade the essential amino acid, l-tryptophan (Trp), along the kynurenine pathway. The two enzymes share a similar active site structure and an analogous catalytic mechanism, but they exhibit a variety of distinct functional properties. Here we used carbon monoxide (CO) as a structural probe to interrogate how the functionalities of the two enzymes are encoded in their structures. With X-ray crystallography, we detected an unexpected photochemical intermediate trapped in a crystal of the hIDO1-CO-Trp complex, where CO is photolyzed from the heme iron by X-rays at cryogenic temperatures (100 K). The CO photolysis triggers a large-scale migration of the substrate Trp, as well as the photolyzed CO, from the active site to a temporary binding site, Sa*. It is accompanied by a large conformational change to an active site loop, JK-LoopC, despite the severely restricted protein motion under the frozen conditions, which highlights the remarkable conformational plasticity of the hIDO1 protein. Comparative studies of a crystal of the hTDO-CO-Trp complex show that CO and Trp remain bound in the active site under comparable X-ray illumination, indicating a much more rigid protein architecture. The data offer important new insights into the structure and function relationships of the heme-based dioxygenases and provide new guidelines for structure-based design of inhibitors targeting them.

Virtual Screening for the Identification of Potential Candidate Molecules Against Envelope (E) and Membrane (M) Proteins of SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes COVID-19, a disease currently spreading around the world. Some drugs are underway or being used to combat this disease. Several proteins of the virus can be targeted in therapeutic approaches. Two structural proteins, membrane (M), envelope (E) have critical roles in virus life cycle, such as assembly, budding, envelope formation and pathogenesis. Here, we employed the in silico strategies to identify and evaluate the selected potential compounds against M and E proteins. For this, the structures of proteins were modeled and then several groups of compounds as FDA approved, natural products or under clinical trials were screened from DrugBank and ZINC databases. The selected dockings were analyzed and the ligands with best binding affinity scores were subjected to evaluate drug-likeness and medicinal chemistry friendliness through prediction of ADMET properties. Normal mode analyses were also performed for six selected complexes to explore the collective motions of proteins. Molecular dynamic (MD) simulation was also performed to calculate the stability of two docked protein–ligand complexes. The results revealed that several compounds had high affinity to the proteins along with some acceptable profiles of mobility and deformability, especially, for M protein.

Distinct conformational dynamics and allosteric networks in alpha tryptophan synthase during active catalysis.

Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this working state represents a four to one ratio of the enzyme bound with the indole-3-glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde-3-phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate- and products-bound states to the working state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in the working state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in the working state. As the enzyme transitions from the substrate-bound to the products-bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit.

Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions.

Protein motions play a fundamental role in enzyme catalysis and ligand binding. The relationship between protein motion and function has been extensively investigated in the model enzyme dihydrofolate reductase (DHFR). DHFR is an essential enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate. Numerous experimental and computational methods have been usedto probe the motions of DHFR through the catalytic cycle and to investigate the effect of distal mutations on DHFR motions and ligand binding. These experimental investigations have pushed forward the study of protein motions and their role in protein-ligand interactions. The introduction of mutations distal to the active site has been shown to have profound effects on ligand binding,hydride transfer rates and catalytic efficacy and these changes are captured by enzyme kinetics measurements. Distal mutations have been showntoexert their effects through a network of correlated amino acids and these effects have been investigated by NMR, protein dynamics, and analysis of coupled amino acids. The experimental methods and thefindings that are reviewed here have broad implications for our understanding of enzyme mechanisms, ligand bindingand for the future design and discovery of enzyme inhibitors.

ABCB1/MDR1/P-gp employs an ATP-dependent twist-and-squeeze mechanism to export hydrophobic drugs.

ABCB1, also called MDR1 or P-glycoprotein, exports various hydrophobic compounds and plays an essential role as a protective physiological barrier in several organs, including the brain, testis, and placenta. However, little is known about the structural mechanisms that allow ABCB1 to recognize hydrophobic compounds of diverse structures or the coupling of ATP hydrolysis to uphill substrate export. High-resolution X-ray crystal structures of the pre- and post-transport states and FRET analyses in living cells have revealed that an aromatic hydrophobic network at the top of the inner cavity is key for the conformational change in ABCB1 that is triggered by a hydrophobic substrate. ATP binding, but not hydrolysis, induces a progressive network that results in a twisting motion of the whole protein, squeezing out the substrate directly to the extracellular space. This twist-and-squeeze mechanism by which ABCB1 exports hydrophobic substrates is distinct from those of other transporters.

Impact of intermolecular attraction and repulsion on molecular diffusion and virial coefficients of spheroidal and rod-shaped proteins

Recognition of protein intermolecular interactions is a key factor for the study and manipulation of protein activity in living systems and biotechnology formulations. Experimental parameter that is sensitive to intermolecular interactions is molecular diffusion. This work presents an extension of an earlier work where we proposed the complementary use of pulsed field gradient nuclear magnetic resonance (PFG NMR) and dynamic light scattering (DLS) to study the specific features of intermolecular interactions via protein translational diffusion. PFG NMR methodology characterizes the thermal motion of protein by means of the self-diffusion coefficient and friction formalism. DLS experiments provide the protein collective (or mutual) diffusion coefficient, which evaluates the direct, non-specific intermolecular interactions of proteins by means of virial coefficients. Theoretical analysis of obtained experimental diffusion data is based on the combination of the friction formalism of nonequilibrium thermodynamics (Vink approach) with the Deryaguin-Landau-Verwey-Overbeek (DLVO) theory. To evaluate the electrostatic potential of proteins with different shape - spheroidal alfa-chymotrypsin (ChTr) and the rod-shaped fibrinogen (Fg) we applied the model of “porous” colloid particles. Our analysis has shown that the atypical local decrease of collective diffusion relative to self-diffusion is a consequence of the dominance of van der Waals attraction over electrostatic repulsion for the rod-shaped Fg.

Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover.

The evolution of new protein functions is dependent upon inherent biophysical features of proteins. Whereas, it has been shown that changes in protein dynamics can occur in the course of directed molecular evolution trajectories and contribute to new function, it is not known whether varying protein dynamics modify the course of evolution. We investigate this question using three related ß-lactamases displaying dynamics that differ broadly at the slow timescale that corresponds to catalytic turnover yet have similar fast dynamics, thermal stability, catalytic, and substrate recognition profiles. Introduction of substitutions E104K and G238S, that are known to have a synergistic effect on function in the parent ß-lactamase, showed similar increases in catalytic efficiency toward cefotaxime in the related ß-lactamases. Molecular simulations using Protein Energy Landscape Exploration reveal that this results from stabilizing the catalytically-productive conformations, demonstrating the dominance of the synergistic effect of the E014K and G238S substitutions in vitro in contexts that vary in terms of sequence and dynamics. Furthermore, three rounds of directed molecular evolution demonstrated that known cefotaximase-enhancing mutations were accessible regardless of the differences in dynamics. Interestingly, specific sequence differences between the related ß-lactamases were shown to have a higher effect in evolutionary outcomes than did differences in dynamics. Overall, these ß-lactamase models show tolerance to protein dynamics at the timescale of catalytic turnover in the evolution of a new function.

FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

Fluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these issues, we present here a robust computer program, called FluoSim, which is an interactive simulator of membrane protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. FluoSim integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the localization and intensity of thousands of independent molecules in 2D cellular geometries, providing simulated data directly comparable to actual experiments. FluoSim was thoroughly validated against experimental data obtained on the canonical neurexin-neuroligin adhesion complex at cell–cell contacts. This unified software allows one to model and predict membrane protein dynamics and localization at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments.

The Active Site of a Prototypical “Rigid” Drug Target is Marked by Extensive Conformational Dynamics

AbstractDrug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off‐target effects, information on protein motion is important. Herein, we demonstrate for the prototypical textbook example of an allegedly “rigid protein” that substantial active‐site dynamics have generally remained unrecognized, despite thousands of medicinal‐chemistry studies on this model over decades. Comparing cryogenic X‐ray structures, solid‐state NMR on micro‐crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, we show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational‐exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines.

The Active Site of a Prototypical "Rigid" Drug Target is Marked by Extensive Conformational Dynamics.

Drug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off‐target effects, information on protein motion is important. Herein, we demonstrate for the prototypical textbook example of an allegedly “rigid protein” that substantial active‐site dynamics have generally remained unrecognized, despite thousands of medicinal‐chemistry studies on this model over decades. Comparing cryogenic X‐ray structures, solid‐state NMR on micro‐crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, we show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational‐exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines.

Polarizable and non-polarizable force fields: Protein folding, unfolding, and misfolding.

Molecular dynamics simulations are an invaluable tool to characterize the dynamic motions of proteins in atomistic detail. However, the accuracy of models derived from simulations inevitably relies on the quality of the underlying force field. Here, we present an evaluation of current non-polarizable and polarizable force fields (AMBER ff14SB, CHARMM 36m, GROMOS 54A7, and Drude 2013) based on the long-standing biophysical challenge of protein folding. We quantify the thermodynamics and kinetics of the β-hairpin formation using Markov state models of the fast-folding mini-protein CLN025. Furthermore, we study the (partial) folding dynamics of two more complex systems, a villin headpiece variant and a WW domain. Surprisingly, the polarizable force field in our set, Drude 2013, consistently leads to destabilization of the native state, regardless of the secondary structure element present. All non-polarizable force fields, on the other hand, stably characterize the native state ensembles in most cases even when starting from a partially unfolded conformation. Focusing on CLN025, we find that the conformational space captured with AMBER ff14SB and CHARMM 36m is comparable, but the ensembles from CHARMM 36m simulations are clearly shifted toward disordered conformations. While the AMBER ff14SB ensemble overstabilizes the native fold, CHARMM 36m and GROMOS 54A7 ensembles both agree remarkably well with experimental state populations. In addition, GROMOS 54A7 also reproduces experimental folding times most accurately. Our results further indicate an over-stabilization of helical structures with AMBER ff14SB. Nevertheless, the presented investigations strongly imply that reliable (un)folding dynamics of small proteins can be captured in feasible computational time with current additive force fields.

Erratum: "Edge expansion parallel cascade selection molecular dynamics simulation for investigating large-amplitude collective motions of proteins" [J. Chem. Phys. 152, 225101 (2020)

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Statistical Laws of Protein Motion in Neuronal Dendritic Trees

SummaryAcross their dendritic trees, neurons distribute thousands of protein species that are necessary for maintaining synaptic function and plasticity and that need to be produced continuously and trafficked to their final destination. As each dendritic branchpoint splits the protein flow, increasing branchpoints decreases the total protein number downstream. Consequently, a neuron needs to produce more proteins to maintain a minimal protein number at distal synapses. Combining in vitro experiments and a theoretical framework, we show that proteins that diffuse within the cell plasma membrane are, on average, 35% more effective at reaching downstream locations than proteins that diffuse in the cytoplasm. This advantage emerges from a bias for forward motion at branchpoints when proteins diffuse within the plasma membrane. Using 3D electron microscopy (EM) data, we show that pyramidal branching statistics and the diffusion lengths of common proteins fall into a region that minimizes the overall protein need.

Persistent Protein Motions in a Rugged Energy Landscape Revealed by Normal Mode Ensemble Analysis.

Proteins are allosteric machines that couple motions at distinct, often distant, sites to control biological function. Low-frequency structural vibrations are a mechanism of this long-distance connection and are often used computationally to predict correlations, but experimentally identifying the vibrations associated with specific motions has proved challenging. Spectroscopy is an ideal tool to explore these excitations, but measurements have been largely unable to identify important frequency bands. The result is at odds with some previous calculations and raises the question what methods could successfully characterize protein structural vibrations. Here we show the lack of spectral structure arises in part from the variations in protein structure as the protein samples the energy landscape. However, by averaging over the energy landscape as sampled using an aggregate 18.5 μs of all-atom molecular dynamics simulation of hen egg white lysozyme and normal-mode analyses, we find vibrations with large overlap with functional displacements are surprisingly concentrated in narrow frequency bands. These bands are not apparent in either the ensemble averaged vibrational density of states or isotropic absorption. However, in the case of the ensemble averaged anisotropic absorption, there is persistent spectral structure and overlap between this structure and the functional displacement frequency bands. We systematically lay out heuristics for calculating the spectra robustly, including the need for statistical sampling of the protein and inclusion of adequate water in the spectral calculation. The results show the congested spectrum of these complex molecules obscures important frequency bands associated with function and reveal a method to overcome this congestion by combining structurally sensitive spectroscopy with robust normal mode ensemble analysis.

Protein conformational entropy is not slaved to water

Conformational entropy can be an important element of the thermodynamics of protein functions such as the binding of ligands. The observed role for conformational entropy in modulating molecular recognition by proteins is in opposition to an often-invoked theory for the interaction of protein molecules with solvent water. The “solvent slaving” model predicts that protein motion is strongly coupled to various aspects of water such as bulk solvent viscosity and local hydration shell dynamics. Changes in conformational entropy are manifested in alterations of fast internal side chain motion that is detectable by NMR relaxation. We show here that the fast-internal side chain dynamics of several proteins are unaffected by changes to the hydration layer and bulk water. These observations indicate that the participation of conformational entropy in protein function is not dictated by the interaction of protein molecules and solvent water under the range of conditions normally encountered.

Role of Protein Motions in Catalysis by Formate Dehydrogenase.

We have analyzed the reaction catalyzed by formate dehydrogenase using transition path sampling. This system has recently received experimental attention using infrared spectroscopy and heavy-enzyme studies. Some of the experimental results point to the possible importance of protein motions that are coupled to the chemical step. We found that the residue Val123 that lies behind the nicotinamide ring occasionally comes into van der Waals contact with the acceptor and that in all reactive trajectories, the barrier-crossing event is preceded by this contact, meaning that the motion of Val123 is part of the reaction coordinate. Experimental results have been interpreted with a two-dimensional formula for the chemical rate, which cannot capture effects such as the one we describe.

Protein-Water and Water-Water Long-Time Relaxations in Protein Hydration Water upon Cooling—A Close Look through Density Correlation Functions

We report results on the translational dynamics of the hydration water of the lysozyme protein upon cooling obtained by means of molecular dynamics simulations. The self van Hove functions and the mean square displacements of hydration water show two different temperature activated relaxation mechanisms, determining two dynamic regimes where transient trapping of the molecules is followed by hopping phenomena to allow to the structural relaxations. The two caging and hopping regimes are different in their nature. The low-temperature hopping regime has a time scale of tenths of nanoseconds and a length scale on the order of 2–3 water shells. This is connected to the nearest-neighbours cage effect and restricted to the supercooling, it is absent at high temperature and it is the mechanism to escape from the cage also present in bulk water. The second hopping regime is active at high temperatures, on the nanoseconds time scale and over distances of nanometers. This regime is connected to water displacements driven by the protein motion and it is observed very clearly at high temperatures and for temperatures higher than the protein dynamical transition. Below this temperature, the suppression of protein fluctuations largely increases the time-scale of the protein-related hopping phenomena at least over 100 ns. These protein-related hopping phenomena permit the detection of translational motions of hydration water molecules longly persistent in the hydration shell of the protein.

How fast are the motions of tertiary-structure elements in proteins?

Protein motions occur on multiple time and distance scales. Large-scale motions of protein tertiary-structure elements, i.e., domains, are particularly intriguing as they are essential for the catalytic activity of many enzymes and for the functional cycles of protein machines and motors. Theoretical estimates suggest that domain motions should be very fast, occurring on the nanosecond or microsecond time scales. Indeed, free-energy barriers for domain motions are likely to involve salt bridges, which can break in microseconds. Experimental methods that can directly probe domain motions on fast time scales have appeared only in recent years. This Perspective discusses briefly some of these techniques, including nuclear magnetic resonance and single-molecule fluorescence spectroscopies. We introduce a few recent studies that demonstrate ultrafast domain motions and discuss their potential roles. Particularly surprising is the observation of tertiary-structure element dynamics that are much faster than the functional cycles in some protein machines. These swift motions can be rationalized on a case-by-case basis. For example, fast domain closure in multi-substrate enzymes may be utilized to optimize relative substrate orientation. Whether a large mismatch in time scales of conformational dynamics vs functional cycles is a general design principle in proteins remains to be determined.