The crystallization of membrane-type proteins remains a field of study that is more art than science. Membrane proteins are critical components of many fundamental biological processes such as ion regulation and transport through the cell membrane and catalyzing reactions such as charge separation and conversion of energy. As such they are often the targets of drug treatments and their malfunction has been linked to numerous diseases. Our understanding of such fundamental membrane-associated biological processes at a molecular level is limited due to the lack of knowledge of high-resolution structure of the membrane proteins as obtained from the analysis of crystalline materials by X-ray diffraction, electron microscopy, or solid state NMR. Numerous factors such as protein amphiphilicity, quantity, and non-rigid conformations increase the difficulty of determining suitable crystallization conditions. Two methods exist for the crystallization of membrane proteins. The in-surfo method utilizes detergents to solubilize membrane proteins, thereby creating a solution that can be screened against various precipitants to find crystal-forming conditions. In prior work we have shown that kinetic control over crystal growth can be achieved by variation of the evaporation rate. However, the in-surfo method suffers from limitations such as the choice of detergent for solubilization, denaturation due to the loss of the lateral membrane pressure, and applicability to various proteins. The in-meso method for protein crystallization allows for the crystallization of membrane proteins without their removal from a membrane-like environment. This alleviates concerns for both the solubilization of amphiphilic molecules and denaturation due to the loss of the lateral membrane pressure. However in-meso crystallization requires the homogeneous mixing of two materials with viscosities differing ~30x. For both of these methods, the determination of crystallization conditions is often done using high throughput sparse matrix screening methods. While traditional well plate technology is well established, such systems typically use large and expensive fluid handling equipment, and may be on a scale that is inaccessible given the quantities of protein available. Here we report on the design, fabrication, and testing of a microfluidic device designed for the on-chip formation and screening of in-meso crystallization conditions for membrane-type proteins at volumes of >20 nL per sample. This platform allows for the preparation of multiple mesophase samples at once, while also facilitating high throughput screening of multiple precipitant solutions on a single chip. In addition to screening for appropriate crystallization conditions, independent control over precipitant injection has the potential to allow for kinetic control of crystal growth done in-meso through the use of additives to control the rate of protein diffusion by affecting the curvature of the protein-containing mesophases. We also demonstrate kinetic control for in-surfo crystallization as achieved by the controlled evaporation of solvent within a microfluidic device. This device enables the rapid identification and optimization of crystallization conditions for membrane proteins and other molecules by precise control of the evaporation rate. In this manner we can independently control the rate of supersaturation and ensure a phase transition for every trial. The advantage of controlling the rate of supersaturation independently manifests in the improvement of crystal quality and an increase in crystal size in the case of soluble as well as membrane proteins. In addition, the microfluidic platform requires only ~5nL of sample per trial. These technology will help facilitate the crystallization of membrane proteins by providing multiple methods for crystal growth and easing the sample volumes necessary for screening. The membrane protein bacteriorhodopsin (bR) is our model system.

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