On the Validation of Protein Force Fields Based on Structural Criteria.

Molecular dynamics simulations depend critically on the quality of the force field used to describe the interatomic interactions and the extent to which it has been validated for use in a specific application. Using a curated test set of 52 high-resolution structures, 39 derived from X-ray diffraction and 13 solved using NMR, we consider the extent to which different parameter sets of the GROMOS protein force field can be distinguished based on comparing a range of structural criteria, including the number of backbone hydrogen bonds, the number of native hydrogen bonds, polar and nonpolar solvent-accessible surface area, radius of gyration, the prevalence of secondary structure elements, J-coupling constants, nuclear Overhauser effect (NOE) intensities, positional root-mean-square deviations (RMSD), and the distribution of backbone ϕ and ψ dihedral angles. It is shown that while statistically significant differences between the average values of individual metrics could be detected, these were in general small. Furthermore, improvements in agreement in one metric were often offset by loss of agreement in another. The work establishes a framework and test set against which protein force fields can be validated. It also highlights the danger of inferring the relative quality of a given force field based on a small range of structural properties or small number of proteins.

A cation-π interaction in a transmembrane helix of vacuolar ATPase retains the proton-transporting arginine in a hydrophobic environment.

Vacuolar ATPases are multisubunit protein complexes that are indispensable for acidification and pH homeostasis in a variety of physiological processes in all eukaryotic cells. An arginine residue (Arg735) in transmembrane helix 7 (TM7) of subunit a of the yeast ATPase is known to be essential for proton translocation. However, the specific mechanism of its involvement in proton transport remains to be determined. Arginine residues are usually assumed to "snorkel" toward the protein surface when exposed to a hydrophobic environment. Here, using solution NMR spectroscopy, molecular dynamics simulations, and in vivo yeast assays, we obtained evidence for the formation of a transient, membrane-embedded cation-π interaction in TM7 between Arg735 and two highly conserved nearby aromatic residues, Tyr733 and Trp737 We propose a mechanism by which the transient, membrane-embedded cation-π complex provides the necessary energy to keep the charged side chain of Arg735 within the hydrophobic membrane. Such cation-π interactions may define a general mechanism to retain charged amino acids in a hydrophobic membrane environment.

Saturation Mutagenesis by Efficient Free-Energy Calculation.

Single-point mutations in proteins can greatly influence protein stability, binding affinity, protein function or its expression per se. Here, we present accurate and efficient predictions of the free energy of mutation of amino acids. We divided the complete mutational free energy into an uncharging step, which we approximate by a third-power fitting (TPF) approach, and an annihilation step, which we approximate using the one-step perturbation (OSP) method. As a diverse set of test systems, we computed the solvation free energy of all amino acid side chain analogues and obtained an excellent agreement with thermodynamic integration (TI) data. Moreover, we calculated mutational free energies in model tripeptides and established an efficient protocol involving a single reference state. Again, the approximate methods agreed excellently with the TI references, with a root-mean-square error of only 3.6 kJ/mol over 17 mutations. Our combined TPF+OSP approach does show not only a very good agreement but also a 2-fold higher efficiency than full blown TI calculations.