Biophysics Seminar - 11/09/2005 - 2:30pm - M100 Starling-Loving hall

Modeling Mitochondrial Calcium induced Calcium Release
Balbir Thomas

Crystallization of membrane proteins in lipidic bilayers
L.V. Misquitta, Yohann Misquitta, V. Cherezov, O. Slattery, J.M. Mohan, D. Hart, M. Zhalnina, W.A. Cramer, M. Caffrey

Abstract

The in meso technique of membrane protein crystallization has been successfully used to crystallize several membrane proteins. However, perceived and actual difficulties with this method have led to it not being extensively adopted. Our work has been aimed at streamlining the crystallization of membrane proteins using the in meso method. To this end, robotic systems have been developed to set up screens and visualize the drops that are used for crystallization, making the process less time-consuming, and less material-intensive (1, 2). New monoacylglycerol systems have also been developed to be used as bilayered hosts in which to grow crystals of membrane proteins (3). To date, the lipids used have had chains 16 and 18 carbon atoms long. We hypothesized that a shorter chained lipid producing a thinner bilayer would facilitate the so-called in meso crystallization process. A 14 carbon monoacylglycerol was chosen as the lipid with which to test the proposal. The target lipid was synthesized and its phase behavior was mapped. Crystals of bacteriorhodopsin and the outer membrane vitamin B₁₂ transporter were successfully produced in this system. The latter is the first β-barrel protein to be crystallized by the in meso method.

References

1. Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y.F., and Caffrey, M. (2004). A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Cryst. D60: 1795-1807.
2. Peddi, A., Zheng, Y.F., Cherezov, V., and Caffrey, M. (2004). Automating the dispensing of viscous biomaterials. Proc. IROS 2004, volume 2: 1281-1286.
3. Misquitta, L.V., Misquitta, Y., Cherezov, V., Slattery, O., Mohan, J.M., Hart, D., Zhalnina, M., Cramer, W.A., and Caffrey, M. (2004). Membrane protein crystallization in lipidic mesophases with tailored bilayers. Structure 12: 2113-2124.

Acknowledgements

Supported in part by Science Foundation Ireland (02-IN1-B266), the National Institutes of Health (GM61070, GM075915), and the National Science Foundation (IIS0308078).