This proposal aims to develop a novel technology based upon self-interaction chromatography (SIC) to measure interactions between membrane proteins, and to apply this technology to the preparation of well-ordered crystals of these molecules. This work is part of a long-term effort to develop methods that will facilitate the acquisition of structural information for membrane proteins. Integral membrane proteins mediate some of the most important processes carried out by living cells, and represent approximately 30% of the proteins encoded in the genome. However, they represent fewer than 1 % of the proteins of known structure, largely because of the extreme difficulties they pose in obtaining diffraction quality crystals. Technologies and methodologies that could rationalize and simplify the crystallization of integral membrane proteins would therefore represent a huge boon to structural biology. The second virial coefficient B22 provides a quantifiable measure of """"""""crystallizability,"""""""" and it should be possible to exploit this quantity to rationally screen and optimize conditions for membrane protein crystallization. However, standard technologies for measuring B22 are not adaptable to high-throughput measurements with membrane proteins. SIC offers a means for rapid and efficient B22 measurements of membrane proteins that circumvents the problems associated with the standard technologies.
The specific aims of this project are to optimize the application of SIC technology to integral membrane proteins, and to use virial coefficient measurements obtained by SIC to test hypotheses about the best ways to crystallize membrane proteins and, ultimately, to quickly identify and optimize crystallization conditions for these molecules. Membrane proteins play crucial roles in both maintaining normal cellular function and in a large number of pathologies. They are the targets of huge number of drugs and toxins, and also mediate resistance to many other drugs. High resolution structural information is absolutely required for any in-depth understanding of the basic functioning of these molecules, as well as for the discovery and development of new therapeutic agents.
