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Unfolding, Refolding, and Hydration of Proteins in the Gas Phase

The gas phase is an unusual environment for studies of biomolecules that function in aqueous solution. But the motivation for these studies is to provide a deeper understanding of the intramolecular interactions and hydration interactions that determine the conformations, and ultimately the physiological properties, of these molecules in solution. Studies of unsolvated proteins provide information about their intrinsic intramolecular interactions. This information will facilitate the refinement and testing of the theoretical methods necessary to understand protein conformations and folding processes. Gas-phase studies also offer new opportunities to study hydration interactions. Starting with an unsolvated protein ion in the gas phase, it is possible to hydrate it, one water molecule at a time, and study the hydration process in exquisite detail. ∆H° and ∆S° of hydration, as a function of the number of adsorbed water molecules, can be obtained from these studies, along with information about how hydration affects the conformation. It is not easy to place intact biological molecules into the gas phase. The studies described here were made possible by the recent development of soft desorption and ionization techniques.1,2 Mass spectrometry is now routinely used to determine the molecular weights of large biomolecules. Methods to obtain sequence information by mass spectrometry have been developed, and mass spectrometry may soon be used to help diagnose diseases and design treatment strategies.3 However, in addition to these important analytical applications, the development of soft ionization techniques has opened the door to an examination of the properties of unsolvated biomolecules, and several groups have started to examine the structures of gas-phase protein ions. McLafferty and collaborators have used H/D exchange.4,5 This work revealed the presence of multiple conformations for unsolvated protein ions, and has provided evidence for gas-phase unfolding and refolding processes. However, the interpretation of gas-phase H/D exchange data remains controversial.6 Douglas, Cooks, and their collaborators have used ion beam scattering to measure collision cross sections for unsolvated proteins and found that the cross section increases with increasing charge.7,8 Other methods that have been used in an effort to deduce structural information include measurement of the surface defects generated by high-energy impact of protein ions,9 the modeling of gas-phase basicities,10 and the analysis of fragment relative kinetic energies.11 In the work described here we have used ion mobility measurements, the gas-phase analogue of electrophoresis, to probe the conformations.12-15 The mobility of an ion in the gas phase, how rapidly it moves through a buffer gas under the influence of a weak electric field, depends on the ion’s average collision cross section with the buffer gas. A protein in an unfolded conformation has a large collision cross section, and it can easily be resolved from the folded conformation which has a much smaller cross section. Mobility measurements are also being used by Bowers13 and Clemmer15 and their collaborators. Bowers’ group has focused mainly on studies of small peptides, while Clemmer’s is exploring a combinatorial approach to understanding secondary structure.