Mechanics of thermal adaptation

Somero’s corresponding state principle relates protein enhanced thermal stability with mechanical rigidity. A natural way to test the mechanical stiffness of a protein is to apply a directional force as in single molecule AFM experiments. Recent experiments have been therefore inspired by the possible correlation among the mechanical and thermal stabilities. Unfortunately single molecule experiments lack molecular resolution, and in silico realisation of thermal and mechanical unfolding can provide very useful insights. This is exactly what we have done in a recent work focused on two homologues belonging to the Cold Shock Protein family. Our results show that for these species there is not a correlation among the thermal resistance of the thermophilic Csp and its mechanical stability. The paper is out in JPC Letter [here].

Stay wet, stay stable?

Proteins often host water molecules inside buried cavities or superficial clefts. The presence of these molecules was first resolved via x-ray crystallography and their exchanging dynamics with the external solution was deeply investigated by NMR experiments, the interested readers can dig all the work done by B. Halle and collaborators, see the Halle's web page. Molecular Dynamics simulations also shed light on the molecular mechanisms of this exchange, earlier work by Hummer and Garcia [1] and Sterpone, Ceccarelli and Marchi [2], date back to 2000 or so. It was always questioned the contribution of this set of molecules to the stability of the protein fold. Very recently we decided to tackle the problem and relate it to the issue of protein thermal stability [3]. The starting question was: is the extra stability of a thermophilic protein correlated to its internal hydration. For our study case, the pair of homologous G-domain from the mesophilic E. coli and the hyperthermophilic S. solfataricus, the answer is yes, at least a bit. The manuscript is here.

[1] G. Hummer and A.E. Garcia "Water Penetration and Escape in Proteins". Proteins 2000, 38, 261−272.
[2] F. Sterpone, M. Ceccarelli, M. Marchi, "Dynamics of Hydration in Hen Egg White Lysozyme. J. Mol. Biol. 2001, 311, 409−419.
[3] O. Rahaman, M. Kalimeri, S. Melchionna, J. Henin, F. Sterpone "Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains ", J. Phys. Chem. B 2014 in press.

Coarse-graining can track protein thermal stability?

Molecular simulation based on coarse-grained (CG) models is commonly used to explore large scale molecular motion or folding/unfolding processes. However, it is questionable wether these simplified models are good enough to capture the different thermal-stabilities of proteins.

Here several problems come upfront. Not all CG models are apt to monitor protein unfolding and stability. In some cases external biases are glued on top of the model in order to ensure proteins stability during a simulation; without these biases the structures just do not hold. As consequence the effect of physical/chemical perturbations to the folded structure cannot be appreciated. In other models, the folded state is encoded in native-biases -as in the Go-like model- so one wonder wether the detected unfolding paths that are key to distinguish the stabilities of homologues are "real". Finally, one should always keep in mind that a CG hamiltonian is made up of effective interactions, and, if a rigorous bottom-up graining approach is used, this implies that the model is temperature and concentration and pressure dependent. That is, the thermodynamic reference state is embedded in the interactions. This makes troubles when simulations are performed at different temperatures as in the case of the Replica Exchange Method. Despite the limitations, the possibility to use a CG model to explore the kinetic and thermodynamic stabilities of mesophilic and thermophilic proteins is rather appealing. We have tackled the problem recently, and nice results have been obtained. The paper is out here.  If you have not access, just ask!

Tracking the corresponding state principle

Maria KALIMERI just graduated from Univ Paris VII. Congratulations! Her thesis focused on mechanical properties of thermophilic proteins explored via an innovative framework based on network analysis. Here a bite of her work "Understanding the relation between protein flexibility, stability and function remains one of the most challenging, open questions in biophysical chemistry. For example, proteins need to be flexible to facilitate substrate binding but locally rigid to sustain substrate specificity. Exemplary cases are enzymes from microorganisms that thrive at elevated temperatures, also referred to as thermophiles. These proteins are stable and functional at the high temperature regime but generally lack activity at ambient conditions. Therefore, their thermal stability has been correlated to enhanced mechanical rigidity through the corresponding states paradigm. The generality of this view, however, has been questioned by a number of

experimental and computational studies. In the present study, we employ the gold standard of computational techniques, namely Molecular Dynamics simulations, in order to identify microscopical characteristics that distinguish thermophilic from mesophilic proteins, elaborating in particular on the rigidity paradigm mentioned above." For a more complete reading see here.

Stay flexible, Stay stable...

For some time thermophilic proteins have been considered more rigid than their mesophilic homologues. The "rigidity paradigm" was introduce to explain both the extreme stability of thermophiles as well as their lack of activity at ambient conditions. According to this view functionality is then recovered at the high optimal growth temperature because of the activation of the protein flexibility. Experimentally, one of the strongest support to this "corresponding states" picture comes from H/D exchange experiments, see this beautiful comment from R. Jaenicke in PNAS(2000). However, recent works using the H/D exchange, Neutron Scattering and NMR techniques have questioned the paradigm. Inspired by this querelle we have used extensive MD simulations to play with the concept for a model system, a pair of homologous G-domains of different stability content. The paper is just out in JPCB(2013). Quite surprisingly for our system we see that the hyperthermophilic protein show comparable and even enhanced flexibility than the mesophilic less stable variant. The more intriguing feature pops up when the global flexibility is considered. The conformational spaces sampled by the proteins were projected on a reduced network of linked kinetic separated states; and the hyperthermophiles is systematically characterised by a larger number of conformational substates! This flexibility (conformational entropy) is proposed to stabilise the protein by broadening the stability curve and consequently raising the melting temperature. I will get back on this........

Graphical Abstract, JPCB (2013), 117 (44), pp 13775–13785. 

Nobel 2013, three legs better than two(?)

The 2013 Nobel prize in Chemistry was a great news for all of us working in the field of computer modeling of biomolecules. M. Karplus, M.Levitt and A. Warshel were recognized for their seminal work on multi-scale modeling of bio-systems, namely for having posed the basis of mixed quantum/classical simulations. However, I like to think the award in a more broad sense, modern science is not anymore solely a duet between experiments and theory. Computation is up there as the third leg of knowledge, a new world with its algorithms, its theory and its in silico experiments. On this regards,  I remember a nice discussion by G. Ciccotti on the role of computing in modern theoretical physics, see here.

Back to the topic of this blog, I just wanted to cite three works from the Nobel’s laureates that are especially important when investigating protein thermostability. The first is a work from V.Daggett and M.Levitt (J.Mol.Biol.1993, see here) where the unfolding pathway of a globular protein is explored by performing high-temperature simulations. The second one is from T.Lazaridis, I. Lee and M. Karplus (Protein Sci. 1997, see here)  where the stability of the hyperthermophilic protein Rubredoxin from Pyrococcus furiosus is compared to that of the mesophilic homologue and discussed vis-à-vis of protein rigidity and flexibility. The final one is from M. Roca, H.Liu, Messer and A. Warshel (Biochemistry, 2007, see here). Here, the authors tackle the problem of protein function at high temperature, and in particular they challenge the common view according to which the lack of activity of thermophiles at ambient conditions relates to a more rigid behavior of the protein.

The first Molecular Dynamics simulation of hard-spheres run on a Univac calculator (BJ Alder and TE Wainwright, JCP, 1957, 27, 1208).  For historical curiosity, see the interview to BJ Alder here and an overview by WW Wood on Monte Carlo methods, here.