Pulsed-Laser Dissociation for Rapid Structural Characterization of Macromolecular Assemblies

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

Genomic and proteomic studies have yielded tremendous insights into the proteins expressed by organisms, but the majority of proteins exist in complexes with other proteins and biomolecules and relatively little is known about these interactions. We have developed a hybrid experimental and theoretical approach to rapidly generate three dimensional models of large macromolecular complexes. We propose to develop new experimental techniques using lasers and mass spectrometry to (1) enable the application of these hybrid structural approaches to additional complexes that are incompatible with the our current methodology and (2) generate additional data that will significantly improve the quality of the resulting three dimensional models. The development of these experiments will significantly improve our ability to characterize the structures of macromolecular complexes. Moreover since complexes serve critical functions in vivo such knowlesge will ultimately affect human health.

Technical Summary

Our overall goal is to determine models of protein complexes, particularly those that are either too dynamic, cannot be reconstituted in vitro or present only at low copy numbers in the cell. This can be achieved at various levels depending on the extent of information available. For example if the overall topology is defined by electron microscopy density then the subunit architecture derived from our experiments can be fitted to provide topological models. If sufficient homology exists then atomic models of protein complexes can be assembled. The majority of complexes that we will study are those that are typically resistant to traditional structural biology methods such X- ray crystallography. To achieve this aim, we have developed a hybrid approach to generate three dimensional models of large macromolecular complexes that use data from a variety of sources, including computational chemistry, proteomics, and mass spectrometry of intact complexes, subcomplexes, and subunits. We propose to take this approach to the next level by developing new gas-phase dissociation techniques using lasers to enable us to achieve higher resolution mass spectra of intact complexes (ie those that are devoid of multiple adducts). We also plan to use this approach to generate extensive fragmentation of intact complexes to fill gaps in our knowledge of the subunits and interaction partners present in intact complexes.

Publications


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