Mapping Protein-Protein Interfaces by MALDI Mass Spectrometry

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Komives9808286Experiments have recently been performed that demonstate the feasibility of measuring hydrogen/deuterium exchange rates of amide protons using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The method permits measurement of the rate of exchange of the very rapidly exchanging amide protons on the surface of proteins. Preliminary results show that the exchange rates of the rapidly exchanging amides decrease if the amides are at the interface in a protein-protein interaction, presumably due to solvent exclusion. In this way, the protein-protein interface can be rapidly mapped. Much work remains to be done to generalize and quantify the method as well as to understand how solvent accessibility relates to protein-protein interaction interfaces. The experimental plan requires first that variables that affect the degree of "coverage" of the protein surface and the effect of analysis time on deuterium retention be determined. Second, the parameters that relate the rates of off-exchange in the complex to the protein concentrations and binding affinity shall be determined. Third, computational methods shall be implemented to ascertain the number of deuteriums on each fragment so that accurate kinetics can be obtained. Fourth, rapid mixing quench methods to monitor conformational changes upon protein-protein complexation will be developed. This experiment should yield a "movie" of the conformational changes occurring upon complexation.This novel method has tremendous potential impact for discovering the functionally important regions of proteins for which structures of their complexes are not available. The interacting interfaces of new proteins identified by the two-hybrid screen could be rapidly identified. It could also prove useful in culling genome information down to a workable size. More importantly, the method provides access to information about the dynamics of protein-protein interfaces. How accessible to the solvent is the protein-protein interface? Does the solvent accessibility change that occurs at the interface depend only on the surface area, or does it also depend on the hydrophobicity of the interface? Do certain regions of the interface become less conformationally flexible upon protein-protein interaction? The MALDI-TOF MS method shall be used to answer these questions on a variety of diverse protein-protein interactions so that the biophysical parameters that govern protein-protein interactions will be better understood.