Innovation technology for membrane protein crystallization


During the past 3 decades,Lasalde_Stevens_Guillermo several research groups have unsuccessfully attempted to obtain a nAChR X-ray structure. During this period we have been developing conditions to prepare high-quality nAChR crystals from Torpedo californica electric tissue. In our recent study (Asmar Rovira et al, 2008) in collaboration with Dr. Raymond C. Stevens at the Scripps Research Institute (La Jolla, CA), we found that during solubilization, a detergent may selectively remove a native lipid specie(s) present in the native cell membrane that could be essential for protein function or stability. This detergent-specific lipid depletion could induce a partial denaturation of the membrane protein hydrophobic domains which could eventually lead to destabilization and premature aggregation. The physico-chemical properties (hydrophobic tail, head-group, CMC, aggregation number, and micelle shape) of the detergent may be critical to lipid exclusion, and the patterns observed in a model membrane protein such as the nAChR could be used to develop correlations in other structurally related membrane proteins. This study also showed that lipid-analog detergents induce a functional stabilization of the nAChR. This lipid-based analysis of the detergent-solubilized nAChR has revealed an innovative perspective on the preparation of high quality nAChR crystals. This lipid-based analysis of the nAChR-detergent complex has revealed an innovative perspective towards the preparation of high quality nAChR crystals. For the first time we propose to perform biophysical studies of the nAChR in the LCP. Our preliminary data demonstrated that the LCP in combination with the lipid based approach could lead to high quality nAChR crystals. This project is technically significant because it will concurrently and systematically assess the lipid composition, ion channel function, and stability (i.e., state of aggregation) of the nAChR- detergent complex and it is innovative in its use of the (LCP-FRAP) approaches in the LCP to estimate nAChR mobile fraction and diffusion coefficient to correlate with receptor stability and/or aggregation. Our research plan will serve to test new approaches in a very challenging project, however, if successful, it will represent a step forward in the use of innovative approaches in other membrane proteins.

Potential nAChR crystals formed in the LCP from LFC-16 solubilized T. californica nAChR .

Video shows the first 85 seconds of a typical LCP-FRAP experiment performed in our laboratory

Fractional fluorescence recovery of each affinity-purified nAChR from Torpedo californica electric organ using fatty acid analog detergent.