The number of integral membrane protein structures determined by x-ray crystallography is growing exponentially. However, the number of structures is approximately equal to that of soluble proteins twenty five years ago. Moreover, determination of membrane protein structures remains, frankly, too difficult and essentially beyond the reach of all but the largest and most exceedingly well-funded laboratories. The overarching Aims of this proposal are to reduce greatly the difficulty, risk and cost of membrane protein structural biology (via x-ray crystallography). This proposal focuses upon the critical testing of several hypotheses; and upon the development of a novel unified technology to aid membrane protein purification, crystallization and structure determination (that can be used on soluble proteins as well).
Specific Aims i nclude: (1) Does modification of surface residues by reductive methylation or surface mutagenesis improve the likelihood of 'successful' crystallization?; (2) Is careful characterization of the detergent and lipid present in purified membrane protein solutions of utility for obtaining well-ordered crystals suitable for structure determination?; (3) Are there any properties of purified membrane protein solutions that are correlative (or even predictive) for formation of well-ordered crystals suitable for structure determination?; (4) Develop a unified novel platform technology based upon the inclusion of optically-active lanthanide-containing domains in proteins or protein-protein complexes. Successful development of this approach will enable: simple optical detection of protein expression, purification and crystallization; fast single-step affinity purification; inclusion of domains to facilitate crystal lattice formation; and inclusion of anomalous scatterers so that phasing is straightforward. The targets for these specific Aims are a range of proteins currently under study (or planned for study) in my laboratory, including the E. coli water channel Aquaporin Z (AqpZ), the E. coli outer membrane cobalamin transporter BtuB, the human plasma membrane copper transporter CTR1, the human mitochondrial peripheral benzodiazepine receptor PBR, and Mycobacterium tuberculosis membrane proteins. Other planned targets will focus upon other human membrane proteins of fundamental and biomedical import. ? ? ? ?
| Vergis, James M; Wiener, Michael C (2011) The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal. Protein Expr Purif 78:139-42 |
| Vergis, James M; Purdy, Michael D; Wiener, Michael C (2010) A high-throughput differential filtration assay to screen and select detergents for membrane proteins. Anal Biochem 407:1-11 |
