Calculation of protein ionization equilibria with conformational sampling: pK_a of a model leucine zipper, GCN4 and barnase

Abstract

The use of conformational ensembles provided by nuclear magnetic resonance (NMR) experiments or generated by molecular dynamics (MD) simulations has been regarded as a useful approach to account for protein motions in the context of pK(a) calculations, yet the idea has been tested occasionally. This is the first report of systematic comparison of pK(a) estimates computed from long multiple MD simulations and NMR ensembles. As model systems, a synthetic leucine zipper, the naturally occurring coiled coil GCN4, and barnase were used. A variety of conformational averaging and titration curve-averaging techniques, or combination thereof, was adopted and/or modified to investigate the effect of extensive global conformational sampling on the accuracy of pK(a) calculations. Clustering of coordinates is proposed as an approach to reduce the vast diversity of MD ensembles to a few structures representative of the average electrostatic properties of the system in solution. Remarkable improvement of the accuracy of pK(a) predictions was achieved by the use of multiple MD simulations. By using multiple trajectories the absolute error in pK(a) predictions for the model leucine zipper was reduced to as low as approximately 0.25 pK(a) units. The validity, advantages, and limitations of explicit conformational sampling by MD, compared with the use of an average structure and a high internal protein dielectric value as means to improve the accuracy of pK(a) calculations, are discussed.

Defining the Role of Salt Bridges in Protein Stability

“…Various protocols combining calculations of ionization equilibria with simulated protein flexibility have been elaborated. In different approaches, the considered structural changes rank from involving only polar hydrogen atoms (18,19,30,33), to side-chain fluctuations (31,32,34,35,38), to global structural motions (6,23,28,(39)(40)(41)(42). Overall, pK values from MD ensemble averages were closer to the experimental values in most cases.…”

“…Multiple shorter MD trajectories (starting from the same initial structure) might be superior to a single MD trajectory of the same total length (41). (iii) The initial structure is likely to influence the results of continuum pK predictions (23), although this is not always the case (43).…”

Salt Bridges and Conformational Flexibility: Effect on Protein Stability

“…We will show below that protein permittivity cannot be used as parameter at ΔG el calculations, either. An alternative approach is to collect conformations using simulation methods such as molecular dynamics [3,5,9,14,22,26,27,28].…”

A comparison of different initialization protocols to obtain statistically independent molecular dynamics simulations

“…A few studies have also tested to increase the sampling by starting from different structures, e.g. from different crystal structures or NMR ensembles [17,25,26,27,28], or even from structures generated by an initial conformational search [25]. Recently, we have also shown that the agreement of calculated and measured order parameters is improved if the MD simulations are started from different structures obtained from conformational disorder available in the crystal structures [18].…”