An algorithm is proposed that generates a set of temperatures for use in parallel tempering simulations (also known as temperature-replica exchange molecular dynamics simulations) of proteins to obtain a desired exchange probability Pdes. The input consists of the number of protein atoms and water molecules in the system, information about the use of constraints and virtual sites and the lower temperature limits. The temperatures generated yield probabilities which are very close to Pdes (correlation 97%), independent of force field and over a wide temperature range. To facilitate its use, the algorithm has been implemented as a web server at .

During electrospray ionization (ESI), proteins are transferred from solution into vacuum, a process that influences the conformation of the protein. Exactly how much the conformation changes due to the dehydration process, and in what way, is difficult to determine experimentally. The aim of this study is therefore to monitor what happens to protein structures as the surrounding waters gradually evaporate, using computer simulations of the transition of proteins from water to vacuum. Five different proteins have been simulated with water shells of varying thickness, enabling us to mimic the entire dehydration process. We find that all protein structures are affected, at least to some extent, by the transfer but that the major features are preserved. A water shell with a thickness of roughly two molecules is enough to emulate bulk water and to largely maintain the solution phase structure. The conformations obtained in vacuum are quite similar and make up an ensemble which differs from the structure obtained by experimental means, and from the solution phase structure as found in simulations. Dehydration forces the protein to make more intramolecular hydrogen bonds, at the expense of exposing more hydrophobic area (to vacuum). Native hydrogen bonds usually persist in vacuum, yielding an easy route to refolding upon rehydration. The findings presented here are promising for future bio-imaging experiments with X-ray free electron lasers, and they strongly support the validity of mass spectrometry experiments for studies of intra- and intermolecular interactions.

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Electrospray ionization is a gentle method for sample delivery, routinely used in gas-phase studies of proteins. It is crucial for structural investigations that the protein structure is preserved, and a good understanding of how structure is affected by the transition to the gas phase is needed for the tuning of experiments to meet that requirement. Small amounts of residual solvent have been shown to protect the protein, but temperature is important too, although it is not well understood how the latter affects structural details. Using molecular dynamics we have simulated four sparingly hydrated globular proteins (Trp-cage; Ctf, a C-terminal fragment of a bacterial ribosomal protein; ubiquitin; and lysozyme) in vacuum starting at temperatures ranging from 225 K to 425 K. For three of the proteins, our simulations show that a water layer corresponding to 3 A preserves the protein structure in vacuum, up to starting temperatures of 425 K. Only Ctf shows minor secondary structural changes at lower starting temperatures. The structural conservation stems mainly from interactions with the surrounding water. Temperature scales in simulations are not directly translatable into experiments, but the wide temperature range in which we find the proteins to be stable is reassuring for the success of future single particle imaging experiments. The water molecules aggregate in clusters and form patterns on the protein surface, maintaining a reproducible hydrogen bonding network. The simulations were performed mainly using OPLS-AA/L, with cross checks using AMBER03 and GROMOS96 53a6. Only minor differences between the results from the three different force fields were observed.

Molecular dynamics simulations of zwitterions of the Trp-cage protein in the gas phase show that the most stable ion in vacuo has preserved the charge locations acquired in solution. A direct comparison of the gas and solution-phase structures reveals that, despite the similarity in charge location, there is significant difference in the structures, with a substantial increase in hydrogen bonds and exposure of hydrophobic parts in the gas phase. The structure of the salt bridge in the gas phase is also much more stable than in the (experimental) solution structure.