Patrice Delarue

How main-chains of proteins explore the free-energy landscape in native states

Understanding how a single native protein diffuses on its free-energy landscape is essential to understand protein kinetics and function. The dynamics of a protein is complex, with multiple relaxation times reflecting a hierarchical free-energy landscape. Using all-atom molecular dynamics simulations of an α/β protein (crambin) and a β-sheet polypeptide (BS2) in their “native” states, we demonstrate that the mean-square displacement of dihedral angles, defined by 4 successive Cα atoms, increases as a power law of time, tα, with an exponent α between 0.08 and 0.39 (α = 1 corresponds to Brownian diffusion), at 300 K. Residues with low exponents are located mainly in well-defined secondary elements and adopt 1 conformational substate. Residues with high exponents are found in loops/turns and chain ends and exist in multiple conformational substates, i.e., they move on multiple-minima free-energy profiles.

Nonexponential decay of internal rotational correlation functions of native proteins and self-similar structural fluctuations

Structural fluctuations of a protein are essential for the function of native proteins and for protein folding. To understand how the main chain in the native state of a protein fluctuates on different time scales, we examined the rotational correlation functions (RCFs), C(t), of the backbone N-H bonds and of the dihedral angles γ built on four consecutive Cα atoms. Using molecular dynamics simulations of a model α/β protein (VA3) in its native state, we demonstrate that these RCFs decay as stretched exponentials, ln[C(t)] ≈ Dαtα with a constant Dα and an exponent α (0 < α < 0.35) varying with the free-energy profiles (FEPs) along the amino acid sequence. The probability distributions of the fluctuations of the main chain computed at short time scale (1 ps) were identical to those computed at large time scale (1 ns) if the time is rescaled by a factor depending on α < 1. This self-similar property and the nonexponential decays (α ≠ 1) of the RCFs are described by a rotational diffusion equation with a time-dependent diffusion coefficient D(t) = αDαtα-1. The present findings agree with observations of subdiffusion (α < 1) of fluorescent probes within a protein molecule. The subdiffusion of 15N-H bonds did not affect the value of the order parameter S2 extracted from the NMR relaxation data by assuming normal diffusion (α = 1) of 15N-H bonds on a nanosecond time scale. However, we found that the RCF does not converge to S2 on the nanosecond time scale for residues with multiple-minima FEPs.

Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins.

Anomalous diffusion and dynamical correlation between the side chains and the main chain of proteins in their native state

Structural fluctuations of a protein are essential for a protein to function and fold. By using molecular dynamics (MD) simulations of the model α/β protein VA3 in its native state, the coupling between the main-chain (MC) motions [represented by coarse-grained dihedral angles (CGDAs) γn based on four successive Cα atoms (n - 1, n, n + 1, n + 2) along the amino acid sequence] and its side-chain (SC) motions [represented by CGDAs δn formed by the virtual bond joining two consecutive Cα atoms (n, n + 1) and the bonds joining these Cα atoms to their respective Cβ atoms] was analyzed. The motions of SCs (δn) and MC (γn) over time occur on similar free-energy profiles and were found to be subdiffusive. The fluctuations of the SCs (δn) and those of the MC (γn) are generally poorly correlated on a ps time-scale with a correlation increasing with time to reach a maximum value at about 10 ns. This maximum value is close to the correlation between the δn(t) and γn(t) time-series extracted from the entire duration of the MD runs (400 ns) and varies significantly along the amino acid sequence. High correlations between the SC and MC motions [δ(t) and γ(t) time-series] were found only in flexible regions of the protein for a few residues which contribute the most to the slowest collective modes of the molecule. These results are a possible indication of the role of the flexible regions of proteins for the biological function and folding.

Low-Frequency, Functional, Modes of Proteins: All-Atom and Coarse-Grained Normal Mode Analysis

The directions of the largest thermal fluctuations of the structure of a protein in its native state are the directions of its low-frequency modes (below 1 THZ), named acoustical modes by analogy with the acoustical phonons of a material. The acoustical modes of a protein assist its conformational changes and are related to its biological functions. Low-frequency modes are difficult to detect experimentally. A survey of experimental data of low-frequency modes of proteins is presented. Theoretical approaches, based on normal mode analysis, are of first interest to understand the role of the low-frequency modes in proteins. In this chapter, the fundamentals of normal mode analysis using all-atom models and coarse-grained elastic models are reviewed. They are applied to proteins intimately related to human diseases: ubiquitin and the 70kDa Heat-Shock Protein (Hsp70). The ubiquitin protein is a single domain protein which is a benchmark for biomolecular Nuclear Magnetic Resonance spectroscopy. Present all-atom calculations predict a “boson peak” near 20 cm− 1 in the inelastic neutron scattering spectra of this protein. The molecular chaperone Hsp70 is an exemplary model to illustrate the different properties of the low-frequency modes of a multi-domain protein, which occurs in two well distinct structural states (open and closed states). The role of the low-frequency modes in the transition between the two states of Hsp70 is analyzed in detail. It is shown that the low-frequency modes provide an easy means of communication between protein domains separated by a large distance.

From a Highly Disordered to a Metastable State: Uncovering Insights of α-Synuclein

α-Synuclein (αS) is a major constituent of Lewy bodies, the insoluble aggregates that are the hallmark of one of the most prevalent neurodegenerative disorders, Parkinsons disease (PD). The vast majority of experiments in vitro and in vivo provide extensive evidence that a disordered monomeric form is the predominant state of αS in water solution, and it undergoes a large-scale disorder-to-helix transition upon binding to vesicles of different types. Recently, another form, tetrameric, of αS with a stable helical structure was identified experimentally. It has been shown that a dynamic intracellular population of metastable αS tetramers and monomers coexists normally; and the tetramer plays an essential role in maintaining αS homeostasis. Therefore, it is of interest to know whether the tetramer can serve as a means of preventing or delaying the start of PD. Before answering this very important question, it is, first, necessary to find out, on an atomistic level, a correlation between tetramers and monomers; what mediates tetramer formation and what makes a tetramer stable. We address these questions here by investigating both monomeric and tetrameric forms of αS. In particular, by examining correlations between the motions of the side chains and the main chain, steric parameters along the amino-acid sequence, and one- and two-dimensional free-energy landscapes along the coarse-grained dihedral angles γ and δ and principal components, respectively, in monomeric and tetrameric αS, we were able to shed light on a fundamental relationship between monomers and tetramers, and the key residues involved in mediating formation of a tetramer. Also, the reasons for the stability of tetrameric αS and inability of monomeric αS to fold are elucidated here.

Characterization of a Drosophila glutathione transferase involved in isothiocyanate detoxification

Glutathione transferases (GSTs) are ubiquitous key enzymes that catalyse the conjugation of glutathione to xenobiotic compounds in the detoxification process. GSTs have been proposed to play a dual role in the signal termination of insect chemodetection by modifying odorant and tasting molecules and by protecting the chemosensory system. Among the 40 GSTs identified in Drosophila melanogaster, the Delta and Epsilon groups are insect-specific. GSTs Delta and Epsilon may have evolved to serve in detoxification, and have been associated with insecticide resistance. Here, we report the heterologous expression and purification of the D. melanogaster GST Delta 2 (GSTD2). We investigated the capacity of GSTD2 to bind tasting molecules. Among them, we found that isothiocyanates (ITC), insecticidal compounds naturally present in cruciferous plant and perceived as bitter, are good substrates for GSTD2. The X-ray structure of GSTD2 was solved, showing the absence of the classical Ser catalytic residue, conserved in the Delta and Epsilon GSTs. Using molecular dynamics, the interaction of ITC with the GSTD2 three-dimensional structure is analysed and discussed. These findings allow us to consider a biological role for GSTD2 in chemoperception, considering GSTD2 expression in the chemosensory organs and the potential consequences of insect exposure to ITC.

Intrinsic Localized Modes in Proteins.

Protein dynamics is essential for proteins to function. Here we predicted the existence of rare, large nonlinear excitations, termed intrinsic localized modes (ILMs), of the main chain of proteins based on all-atom molecular dynamics simulations of two fast-folder proteins and of a rigid α/β protein at 300 K and at 380 K in solution. These nonlinear excitations arise from the anharmonicity of the protein dynamics. The ILMs were detected by computing the Shannon entropy of the protein main-chain fluctuations. In the non-native state (significantly explored at 380 K), the probability of their excitation was increased by a factor between 9 and 28 for the fast-folder proteins and by a factor 2 for the rigid protein. This enhancement in the non-native state was due to glycine, as demonstrated by simulations in which glycine was mutated to alanine. These ILMs might play a functional role in the flexible regions of proteins and in proteins in a non-native state (i.e. misfolded or unfolded states).

Data on the expression of GSTE1 and GSTE7 in Drosophila chemosensory organs after isothiocyanate exposure

The data presented in this article are related to the research article entitled “Characterization of a Drosophila glutathione transferase involved in isothiocyanate detoxification.” (Gonzalez et al., 2018) [1]. This article includes the expression level of Drosophila melanogaster GSTE1 and GSTE7 in chemosensory male tissues and the expression level of the mRNAs coding for the same enzymes after a PEITC exposure in food.

Fingerprints of Conformational States of Human Hsp70 at Sub-THz Frequencies

Large multidomain proteins occur in different conformational states to function. Detection and monitoring of these different structural states are of crucial interest for understanding the mechanics of proteins. Using computational methods, we show that different protein conformational states of the two-domain 70 kDa human Heat-shock protein (hHsp70), with similar vibrational density of states, lead to remarkably different far-IR spectra at acoustical frequencies (ν < 300 GHz). We found that the slow damped motions of the positively charged residues of hHsp70 contribute the most to collective IR active modes at low frequencies (ν < 300 GHz). We predicted that different structural states and functional modes of large proteins, such as hHsp70, might be detected in the sub-THz frequency range by single-molecule spectroscopy similar to the recent extraordinary acoustic Raman spectroscopy (WheatonS.; Nat. Photonics2015, 9, 68−72).