(606a) High-Throughput Membrane Protein Crystallization Condition Screening Using X-Ray Transparent In Meso Microfluidic Chips

The malfunction of membrane proteins has been tied to the initiation and progression of many diseases and, consequentially, membrane proteins account for ~60% of all available drug targets.  High-resolution 3-D structures of proteins are essential to understand the structure-function relationship of these molecules and the changes therein that contribute to disease states.  However, the amphipathic nature of membrane proteins, the difficulty of expressing and purifying them, and their low yield upon purification have all made them very difficult to successfully screen and crystallize.  An emerging technique called the in mesomethod has increased success rates of membrane protein crystallization.  This method suffers from limited adoption because of a variety of factors such as the requirement of specialized equipment, expert labor, or expensive robots.  Upon successful crystallization, the next bottleneck is the manual handling of the fragile crystals while mounting them on a loop or a capillary, and subsequent cryo-cooling to collect X-ray diffraction data – a process where many crystals suffer damage resulting in poor diffraction data.

To address these challenges we have developed high-throughput X-ray transparent microfluidic chips for in meso crystallization screening.  The chips are designed with 12-, 24-, and 48- well arrays requiring < 20 nL of protein solution per well and also simultaneously automate the formulation of each sample.  X-ray transparency allows these microfluidic chips to circumvent the need for manual crystal harvesting, thus preserving the initial quality of crystals.  These in meso chips enable the use of lipidic mesophases for crystallization screening on an X-ray transparent platform, a previously unachieved accomplishment.  Our microfluidic platforms simplify in meso sample preparation and allow for crystallization condition screening, crystal growth and the acquisition of high-resolution diffraction data on chip for structure solution. We have validated our approach on bacterial photosynthetic reaction center and are currently working on challenging membrane proteins from the heme-copper oxidase superfamily.