Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study

Abstract

The proton transfer activity of the light-driven proton pump, bacteriorhodopsin (bR) in the photochemical cycle might imply internal water molecules. The free energy of inserting water molecules in specific sites along the bR transmembrane channel has been calculated using molecular dynamics simulations based on a microscopic model. The existence of internal hydration is related to the free energy change on transfer of a water molecule from bulk solvent into a specific binding site. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each hydrated model from molecular dynamics simulations of the creation of water molecules into specific protein-binding sites. A rigorous statistical mechanical formulation allowing the calculation of the free energy of transfer of water molecules from the bulk to a protein cavity is used to estimate the probabilities of occupancy in the putative bR proton channel. The channel contains a region lined primarily by nonpolar side-chains. Nevertheless, the results indicate that the transfer of four water molecules from bulk water to this apparently hydrophobic region is thermodynamically permitted. The column forms a continuous hydrogen-bonded chain over 12 A between a proton donor, Asp 96, and the retinal Schiff base acceptor. The presence of two water molecules in direct hydrogen-bonding association with the Schiff base is found to be strongly favorable thermodynamically. The implications of these results for the mechanism of proton transfer in bR are discussed.

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

“…When focusing on the average value associated to each pocket, we obtain ⟨ΔG i ⟩ P1 = −1.6 ± 0.3 and ⟨ΔG i ⟩ P2 = −5.5 ± 0.4 kcal/mol for the domain and ⟨ΔG i ⟩ P1 = −1.5 ± 0.3 and ⟨ΔG i ⟩ P2 = −3.6 ± 0.5 kcal/mol for the domain. The values are close to previous estimates of the insertion free energy for water into the cavities of other proteins as the bacteriorhodopsin 48 , BPTI 50 , and kinase A 80 .…”

“…According to the analysis presented in 48,53 , our values correspond to the insertion free energy of a molecule in the presence of n − 1 others. The ensemble of conformers A i , B i , C i helps us to sample several initial conditions of the hydrated cavities, i.e.…”

“…Alternative strategies to estimate the free energy contribution to stability stemming from the hydration of large cavities have been presented before and reporting for non-polar cavities and channels cooperative effects 22,48,53 . For example in hydrophobic pockets of large volumes favorable hydration is found to take place only above a given threshold: about 4/5 water molecules should be hosted to compensate the loss of HBs due to the cavity nature 22 .…”

Structure, dynamics and reactions of protein hydration water

“…Central to this is an understanding of the free energy change on binding, owing to changes in the nature of the degrees of freedom of the ligand. A theoretical framework for determining the free energy of transfer of a water molecule into a given site in a protein has been derived (Roux et al 1996). An example of a wellordered water molecule is found in the BPTI.…”

Comment on “Free energy simulations of single and double ion occupancy in gramicidin A” [J. Chem. Phys. 126, 105103 (2007)]

“…In particular, the accuracy of the atomic force field remains a central issue in ion channel simulations. However, there is no ambiguity about the proper theoretical treatments for calculating equilibrium binding constants, either from alchemical free energy perturbations, [11][12][13][14][15][16][17] or from integration of an unrestricted 18 as well as a restricted 1D PMF. [2][3][4]19 It is only by using unbiased estimators of experimentally observable quantities that one can assess the validity of those simulations.…”