High-throughput crystallization of membrane proteins in lipidic mesophases
Vadim Cherezov, Chemistry Department, The Ohio State University, Columbus, OH 43210
An introduction to the fascinated world of membrane protein crystallization in lipidic mesophases will be given with an emphasis on the high-throughput approach.
Crystallization in lipidic mesophases (in meso) is a relatively new and promising technique. It has proved its success in producing high-resolution structures of bacteriorhodopsin,
including its various mutants and photointermediates and few other mostly rhodopsin-related proteins. Progress has been limited by a poor understanding of the crystallization
mechanism and practical difficulties in handling very viscous lipid/protein mixtures. We have put forward an hypothesis for how the crystallization occurs and are currently in the
process of testing them using microfocus x-ray diffraction and small-angle neutron scattering. The handling problem was solved by building a high-throughput in meso crystallization
system that includes an in meso crystallization robot, special glass-based crystallization plates and an automatic imager. The in meso robot can set up crystallization in a 96-well
plate within 9 minutes and uses just 20 nL of protein, 30 nL of lipid and 1 mL of precipitant solution per trial. The glass-based plates provide unsurpassed optical birefringence-free
properties for reliable screening of very small micron-size crystals of colorless proteins grown in lipidic mesophases. The automatic imager was designed to obtain the best possible
images of the in meso crystallization bolus. A 96-well plate can be imaged in 4.5 minutes. The robotic system enables high-throughput crystallization trials in lipidic mesophases with
unprecedented speed using minimal quantity of protein. Recent success in obtaining crystals of the outer membrane b-barrel transporter, BtuB, diffracting to 2.5 Å expands the
applicability of the in meso approach to a completely new class of membrane proteins.
Supported in part by Science Foundation Ireland (02-IN1-B266), the National Institutes of Health (GM61070), and the National Science Foundation (DIR9016683 and DBI9981990).
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