Conformations of anhydrous and partially hydrated proteins

Water is of central importance for protein biological activity. The complex interactions of water molecules with a protein stabilize its "native", that is, biologically active, three-dimensional structure. Many proteins exhibit biological activity only in conjunction with cofactors (for example, heme), and also here water molecules effect stabilization of the protein-cofactor complexes to form a biologically active structure. Structural water molecules can even be found at the interface of a protein and its cofactor, in which cases the water can be considered an integral part of the active complex. Only after completion of their biosynthesis at the ribosome, proteins can adopt their functional structure. In this protein folding process, water also plays an essential role that is, however, not yet sufficiently understood. Recently, more and more attention is being directed toward the role of water in protein function, and proteins in the condensed phase are increasingly studied by NMR spectroscopy and X-ray crystallography under the aspect of protein hydration. A fundamentally different and new experimental approach that, in contrast to the above methods, allows for the study of completely desolvated and partially hydrated proteins, will be applied in this project. Proteins will first be liberated from the bulk solvent in a spray process, and remaining water molecules subsequently evaporated in a controlled fashion. Depending on the experimental conditions chosen, completely desolvated or partially hydrated proteins can be generated and studied in a special mass spectrometer with ion storage capabilities. The research approach employs a new mass spectrometry technique, electron capture dissociation (ECD), which permits localization of water molecules within the partially hydrated proteins. Moreover, the structural information provided by ECD can be used to evaluate changes in protein structure upon hydration, and to determine folding energetics of desolvated protein ions. The goal of this project is to obtain experimental answers to some hotly debated current questions: To what extent do gaseous protein ion and protein ion-ligand complex structures resemble their solution counterparts? How does hydration effect the structures of gaseous protein ions and protein ion-ligand complexes? What is the effect of hydration on protein folding energetics? Apart from their fundamental relevance to biochemical processes, the results obtained in this project will be highly important for analytical biochemistry by mass spectrometrometry methods, and they constitute an experimental link to protein model calculations.

The goal of the project P15767 was to "better understand gaseous protein ion conformations...and to comprehend the effect of hydration on protein conformation and folding". The gaseous protein conformers that ultimately form after desolvation and thermal equilibration were studied by electron capture dissociation (ECD) in a Fourier transform - ion cyclotron resonance (FT-ICR) mass spectrometer. Combined ECD, H/D exchange, and infrared photodissociation spectroscopy (IRPDS) data lead to proposed ubiquitin ion gas phase structures for their various charge states observed with electrospray ionization (ESI). The finding of a mostly helical secondary structure in the gaseous 7+ ubiquitin ions from ESI of non-denaturing solutions contradicts the often made assumption that the native protein structure (with 43% ß-sheet and only 21% a-helix content for ubiquitin) is retained in the gas phase. The effect of hydration on protein structure was studied by "native electron capture dissociation" (NECD), a phenomenon newly discovered in the course of this project. NECD occurs at the early stages of protein ion desolvation within the ESI process, and can be used to characterize intermediates in the structural reorganization of proteins during transfer into the gas phase, as well as to obtain site-specific information on the effect of hydration on protein stability. The analysis for equine Cytochrome c showed that the first step in the conformational reorganization as a result of dehydration is the separation of the terminal helices, followed by the unfolding of the His18-loop (residues 18-34), consistent with a substantial weakening of hydrophobic bonding after solvent removal. An analysis of temperature-dependent data gave relative activation energies for the dissociation of individual protein/heme interactions in Cytochrome c. The order of stability was found to be T49 > F82 > L68 > K13, demonstrating the higher stability of hydrogen bonding (T49) over hydrophobic bonding (F82, L68, K13) in a gaseous environment. Thus the most stable structural region in solution, the crossed terminal helices, is the least stable in the gas phase, and the second least stable region in solution comprising the T49/heme interaction is the most stable in the gas phase.