BIOT 140 |
| There are several limiting factors in crystallographic studies of integral membrane proteins – the most difficult to overcome being purification of sufficient quantities of protein and generation of well-ordered crystals. Our research has focused on structural studies of the sarcoplasmic reticulum calcium pump in a variety of inhibitory complexes. Phospholamban (PLB) and sarcolipin (SLN) are small integral membrane proteins that regulate the calcium pumps in cardiac and skeletal muscle, respectively. Defects in regulation by PLB are a central determinant in heart disease and end-stage heart failure. Recent evidence suggests that defects in regulation by SLN may have consequences in the atria of the heart, as well as the neuromusculature. While these regulatory interactions are biochemically and physiologically well characterized, structural details are lacking. To pursue structural studies by electron and X-ray crystallography, large quantities of pure protein are required. Multi-milligram quantities of the calcium pump are purified from native sources using a well-established protocol. PLB and SLN are over-produced in bacterial cell culture as maltose binding protein fusion proteins. A procedure involving protease cleavage, selective solubilization with denaturants, and reverse-phase HPLC permits the rapid, large-scale production of highly pure PLB and SLN protein. From these pure proteins, we have generated well-ordered crystals suitable for high resolution crystallographic studies. The addition of lipids is required, and our biggest surprise has been the dependence of crystal morphology, order and space group on the choice of lipid. Two examples include (i) the addition of phosphatidic acid in the presence of divalent cations generates a novel P22121 crystal form (1), and (ii) the crystalline order can be improved by the addition of small amounts of particular lipids (phosphatidylethanolamine or dioleoylphosphatidylcholine). 1. Stokes, D., Pomfret, A., Rice, W., Glaves, J., and Young, H. (2006) Biophysical Journal 90, (in press).
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Biophysical and Biomolecular Symposium: Challenges to Membrane Protein Production and Characterization
8:00 AM-10:55 AM, Tuesday, 12 September 2006 Hilton San Francisco -- Imperial B Ballroom, Oral
Division of Biochemical Technology |