Elsevier

Journal of Non-Crystalline Solids

Volume 407, 1 January 2015, Pages 494-501
Journal of Non-Crystalline Solids

Are coarse-grained models apt to detect protein thermal stability? The case of OPEP force field

https://doi.org/10.1016/j.jnoncrysol.2014.07.005Get rights and content

Abstract

We present the first investigation of the kinetic and thermodynamic stability of two homologous thermophilic and mesophilic proteins based on the coarse-grained model OPEP. The object of our investigation is a pair of G-domains of relatively large size, 200 amino acids each, with an experimental stability gap of about 40 K. The OPEP force field is able to maintain stable the fold of these relatively large proteins within the hundred-nanosecond time scale without including external constraints. This makes possible to characterize the conformational landscape of the folded protein as well as to explore the unfolding. In agreement with all-atom simulations used as a reference, we show that the conformational landscape of the thermophilic protein is characterized by a larger number of substates with slower dynamics on the network of states and more resilient to temperature increase. Moreover, we verify the stability gap between the two proteins using replica-exchange simulations and estimate a difference between the melting temperatures of about 23 K, in fair agreement with experiment. The detailed investigation of the unfolding thermodynamics allows to gain insight into the mechanism underlying the enhanced stability of the thermophile relating it to a smaller heat capacity of unfolding.

Introduction

Proteins are marginally stable soft-matter entities, with a free energy difference between the folded and the unfolded states of only a few kcal/mol [1]. This small difference, about 3 to 30 kcal/mol, which microscopically corresponds to just a few hydrogen bonds, results from a delicate balance of intramolecular and solvation forces that causes a large enthalpy–entropy compensation.

The design of proteins of enhanced stability is a key goal for many applications in biotechnology and chemical processing aimed at exploiting the catalytic power of enzymes in non-native harsh conditions [2]. In this regard, proteins from thermophilic organisms, which thrive at temperatures as high as the boiling point of water, represent a natural template [2], [3]. Understanding both the thermodynamics as well as the molecular basis of their resistance to high temperatures could be fundamental for de novo protein design.

It is widely accepted that thermophiles gain stability, with respect to their mesophilic homologues that work at ambient conditions, by a combination of molecular factors [2], [4], [5]. The commonly observed surplus of charged amino-acids is for example associated to an extended network of hydrogen bonds (HB) and ion-pairs that eventually rigidify strategic regions of the protein matrix [6], [7] and enhance the coupling with the solvent. In addition, the extension of hydrophobic contacts has been proposed as a source of cohesive forces that stabilize the folded state [8]. Structurally speaking, the shorter loops and flexible regions detected in the structure of thermophilic proteins as well as their distribution along the sequence reduce the number of weak spots on the protein surface preventing unfolding [9]. As a consequence of these and other factors, several thermodynamic mechanisms have been identified as responsible for an enhanced thermal stability [10], [2], [11], [5], [12].

Nowadays, the increase of computational power makes feasible an extended investigation of the kinetic and thermodynamic properties of this class of proteins by computer simulations, although limitations still exist [5]. For example the behavior of homologue proteins can be explored via brute force atomistic molecular dynamics at the microsecond time scale and longer, allowing to compare their folded-state flexibilities or track their different kinetic stabilities at high temperature. Unfortunately, for all atom (AA) models in the explicit water the calculation of thermodynamic properties like the exact melting temperature, the heat capacity of unfolding or the overall shape of stability curves is still a challenge even for medium-size molecules. For that reason the use of coarse-grained (CG) potentials in combination with enhanced-sampling techniques is an appealing alternative [13].

Simplified models have been already successfully applied to the design of proteins of enhanced stability, for example by targeting specific structural patterns [14] or optimizing electrostatic interactions [15]. However, these approaches rely on static protein structures. Accounting for molecular flexibility would open alternative routes for in silico design by including, in some sense, entropic effects and kinetic stabilization. Here we make a first attempt to use a protein CG model in combination with standard MD and an enhanced sampling technique in order to explore the different thermal stabilities of two homologous proteins.

The model recruited for the task is the optimized potential for efficient protein structure prediction (OPEP), a coarse-grained force field designed to fold peptides and small proteins that has already been used successfully in a wide variety of cases (see Refs. [16], [17], [18], [13], [19] and references therein). The model is used here to study the different stabilities of two relatively large homologous proteins, having about 200 amino acids each. The first one is the catalytic d'omain of the elongation factor thermo unstable (EF-Tu) from the bacterium Escherichia coli [20], a mesophile, while the second one is the catalytic domain of the homologous EF-1α from the archaeon Sulfolobus solfataricus, a hyperthermophile [21]. This pair of homologues is a very good study-case for several reasons; their thermal stabilities are separated by a large gap of about 40 K, their fold contains both α and β structures, and the hyperthermophile is enriched in charged amino acids as commonly observed in thermophiles.

We recently studied these two proteins using all-atom molecular dynamics, with which we probed the enhanced stability of the hyperthermophilic variant at high temperature and analyzed the folded state at ambient conditions [22], [23], [9]. This all-atom investigation represents a reference state for benchmarking the capability of the OPEP force field.

Herein, we verify that OPEP can extend standard MD simulations of rather large proteins in the hundred-nanosecond timescale without compromising their overall structures. Furthermore, specific features of the two homologues, such as the different number of sub-states characterizing the conformational landscape or their collective motion, qualitatively reproduce the results from the all-atom simulations. Using this folded-state characterization we provide a first mapping of the OPEP CG time-scale versus the all-atom one for internal protein dynamics. Finally, with the use of replica-exchange molecular dynamics simulations we investigate the different thermal stabilities of the proteins; not only we reproduce the proteins' stability gap, but also gain an insight into the underling thermodynamic mechanism.

Section snippets

Methods

As mentioned briefly above, the two homologous proteins under study are the G-domains of the elongation factor thermo unstable (EF-Tu) and 1α (EF-1α). The mesophilic protein, M (EF-Tu, PDB code 1EFC [21]), belongs to E. coli bacterium while the hyperthermophilic one, H (EF-1α, PDB code 1SKQ [20]), belongs to the S. solfataricus archaeon. The G-domain corresponds to the N-terminal part of the protein. In our simulations the mesophilic homologue covers the residues T8-E203 and its size is 196

Stability on long time scale

So far the OPEP force field has been extensively applied to study small peptides, and it was only recently tested on mid-size proteins with generally less than 80 amino-acids [32], [17], [23]. Therefore, given their size, the mesophilic (M) and hyperthermophilic (H) G-domains are a challenging study-case. The capability of the force field to maintain the fold of the two proteins in MD simulations extending up to 100 ns is first probed and discussed below.

After an equilibration phase at low

Conclusions

This is the first time that the thermal stability of two homologous thermophilic and mesophilic proteins is examined using the OPEP force field. In fact, to the best of our knowledge, this has never been attempted with any other coarse-grained force field of the same nature for such large systems consisting of about 200 residues. First, we show the capability of the force field to preserve the native state at the time scale of a hundred of nanoseconds by MD without the need of external

Acknowledgments

The research leading to these results has received funding from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007-2013) Grant Agreement no. 258748. Figures and calculations were partly done using the R software and packages bio3d and igraph [56], [57], [58]. Part of this work was performed using HPC resources from GENCI [CINES and TGCC] (Grant 2012 c2012086818 and 2013 x201376818). We acknowledge the financial support for infrastructures from

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