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.

Volume fluctuation of reaction intermediates determines reactivity

Fig. 6. in Terazima et al, PNAS (2014) shows that a monoactivated protein decamer expands in volume and consequently dissociates, while a polyactivated decamer shrinks in volume and doesn't dissocitate.

 

Proteins are subjected to thermal fluctuations that occur at all length- and timescales, ranging from bond vibrations to large-scale domain rearrangements. The fluctuations affect the volume of the protein and vice versa. The close relationship between protein conformation and protein activity is a well established concept, while the role of fluctuations in protein activity is yet to be elucidated. A recent experimental study on a blue-light photosensor (TePixD) from the thermophile T. elongatus has shown that the flexibility of reaction intermediates is crucial in the overall reaction kinetics.

In solutions and crystals, TePixD proteins form pentameric rings, which further associate to decameric structures. An equilibrium between the pentamers and decamers is impaired by light illumination, upon which the decamers dissociate to pentamers. If the light-source is then removed, the pentamer-decamer equilibrium is restored within 5 s.

Using a pulse laser-based technique, two intermediate states have been identified in the decamer dissociation upon light illumination. These intermediates are named I1 and I2, and they exhibit different volume compressibilities with respect to the ground state. It has been shown that the change in compressibility, which is proportional to change in volume, is positive and about 10% of the compressibility of the protein, meaning that the intermediate species’ volume is larger as compared to the ground state. The volume change is related to different fluctuation levels. It is presumed that the fluctuations are unequally distributed and that the enhancement of the fluctuations at the interface regions of the decamers facilitates the dissociation.

The light illumination causes a conformational change in the immediate vicinity of the photoreactive center, but this center is not near the protein interfaces that are affected by dissociation and association. Evidently, the protein flexibility is changed by the conformational change around the photoactive center, which causes a greater protein flexibility, which in turn increases the volume of the intermediate species and drives protein dissociation.

This is also confirmed by the fact that high-intensity light, which activates more than one protein in the decamer, produces less pentamers because the volume of these species is decreased as well as the protein fluctuations. Thus, there exists an experimental proof that relates higher protein flexibility to enhanced reactivity.

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!