Microfluidic platforms for membrane protein crystallization and in situ crystallography

Membrane proteins, biological macromolecules that reside in cellular membranes, play critical roles in many biological process, including signaling, transport, and intercellular communication. The malfunction of membrane proteins has been linked to the initiation or progression of many diseases (e.g. autism, diabetes), so the study of their precise structure is of critical interest in the field of drug discovery and structure-based drug design. Structure-based drug development relies on the knowledge of atomic resolution 3D protein structures, the relationship between protein function and structure, and how these proteins interact with potential drug molecules. X ray crystallography, presently the most common and robust method for solving structures, relies on the growth of high quality protein crystals. The structural pipeline reaches a bottleneck during X-ray crystallography because conditions for protein crystallization cannot be determined a priori – extensive screening methods (i.e. trial and error) across a multi-parametric chemical space must be conducted to discover appropriate crystallization conditions using limited amounts of precious membrane protein sample. Analysis of eukaryotic genomes that 30% of all proteins are membrane proteins, however <2% of all known structures are membrane proteins. Given their significant role in disease and the slow pace of structure elucidation, new methods are needed to accelerate structure discovery for membrane proteins. The membrane protein crystallization toolbox contains many powerful, yet difficult-to-use tools. For example, nucleation and growth are typically coupled during crystallization experiments, which limits the degree of control over the quality and size of crystals grown in solution. Seeding techniques, where crystals grow from existing nuclei, provide a straightforward route to large, diffraction quality crystals. While simple in principle, seeding is difficult in practice because the experimental procedure requires the crystallographer to disrupt the equilibrium of the crystallization droplet, and oftentimes ruining the crystallization experiment. This difficulty often leaves seeding as a ‘last resort’ technique. Another technique, in meso crystallization, maintains membrane proteins in a native-like membrane throughout the process of crystallization and has yielded very high quality crystals and structures of previously intractable membrane proteins. Unfortunately, the in meso method requires handling highly viscous lipid phases with specialized mixing and dispensing tools, and is thus limited to dedicated crystallographers and labs with robotic formulation systems. Regardless of the crystallization technique used, membrane protein crystals are incredibly fragile, so the final step of harvesting and mounting crystals prior to X-ray diffraction also hampers progress in structural studies. This dissertation details the development and application of a suite of microfluidic crystallization platforms designed to overcome technical difficulties in membrane protein crystallization. Specifically, these platforms enable crystallization condition screening for either seeding techniques or in meso techniques and subsequent in situ X-ray crystallography. This work improves upon the construction of X-ray transparent devices previously designed in the Kenis group and applies them to membrane protein crystallography. In Chapter 2, devices for separating nucleation and growth via crystal seeding were developed and applied to a model soluble protein and a target membrane protein. In Chapter 3, a novel microfluidic method for formulating in meso crystallization trials was developed and used to crystallize and solve the structure of a membrane protein. In Chapter 4, in meso crystallization devices for high-throughput screening and optimization experiments were designed, and crystallization conditions for several membrane proteins of unknown structure were discovered. In the interest of directly studying protein-ligand or protein-drug interactions, a novel microfluidic method for growing and subsequently soaking crystals in meso was developed and applied to a model crystallization system in Chapter 5. In summary, this work details the development of microfluidic platforms that automate membrane protein crystallization through a variety of techniques. These devices incorporate fine control at the nanoliter scale and in situ analysis into high-throughput arrays to facilitate membrane protein structure determination. The development of platforms for in meso crystallization is particularly significant, as they represent the first X-ray transparent microfluidic platforms for in meso crystallization which also push the limits of scale and throughput when compared to state-of-the-art robotic in meso techniques. Further, when extended to studying protein-ligand and protein-drug systems via soaking, the in meso approach demonstrated here presents an attractive route to develop and study pharmaceuticals

Microfluidic device for super-fast evaluation of membrane protein crystallization

Membrane proteins embedded in bi-layer lipids of cell membrane have unique functions including inter-cell communication, ions/molecules transport. And there is more than 50% of drug design emphasizes on membrane proteins specifically studying on their structure and formation. Recently we reported the structural and functional studies of membrane protein lipid nanoparticles in native biological membrane. This virus-like nanoparticle formed by a self-assembly crystallization process of membrane protein and lipids is critical to pharmaceutical industrial. These nanoparticles have a variety of potential applications in drug delivery and drug design that can carry specific the membrane protein on aim or release control. The previous studies stay on an inefficient method with a standard dialysis process that has low-throughput, time consumption, and protein sample waste. However, the interdisciplinary cooperation between in biology and Micro electro mechanical systems (MEMS) has been tremendous developed such as Bio-MEMS and Lab-on-achip technologies. Here we demonstrate a new concept with a high-throughput membraneless microfluidic device to fast produce the reconstitution of membrane protein nanoparticles. The reconstitution process in continuous micro flow dominated by convection-diffusion phenomena in microfluidic channel can be completed in seconds to form protein/lipid particles under multiple conditions applied. The controllable syringe pumps is used to test a combination of conditions rather than using inefficient hand pipette. Moreover this novel microfluidic device can save protein sample consumption down to only nanoliter or picoliter. By using this device, we have an ability to rapidly form uniform membrane protein lipid nanoparticles and we believe this new method will make a transformative impact to commercial applications in variety of areas from biology to pharmacology

Microfluidic device for super-fast evaluation of membrane protein crystallization

Membrane proteins embedded in bi-layer lipids of cell membrane have unique functions including inter-cell communication, ions/molecules transport. And there is more than 50% of drug design emphasizes on membrane proteins specifically studying on their structure and formation. Recently we reported the structural and functional studies of membrane protein lipid nanoparticles in native biological membrane. This virus-like nanoparticle formed by a self-assembly crystallization process of membrane protein and lipids is critical to pharmaceutical industrial. These nanoparticles have a variety of potential applications in drug delivery and drug design that can carry specific the membrane protein on aim or release control. The previous studies stay on an inefficient method with a standard dialysis process that has low-throughput, time consumption, and protein sample waste. However, the interdisciplinary cooperation between in biology and Micro electro mechanical systems (MEMS) has been tremendous developed such as Bio-MEMS and Lab-on-achip technologies. Here we demonstrate a new concept with a high-throughput membraneless microfluidic device to fast produce the reconstitution of membrane protein nanoparticles. The reconstitution process in continuous micro flow dominated by convection-diffusion phenomena in microfluidic channel can be completed in seconds to form protein/lipid particles under multiple conditions applied. The controllable syringe pumps is used to test a combination of conditions rather than using inefficient hand pipette. Moreover this novel microfluidic device can save protein sample consumption down to only nanoliter or picoliter. By using this device, we have an ability to rapidly form uniform membrane protein lipid nanoparticles and we believe this new method will make a transformative impact to commercial applications in variety of areas from biology to pharmacology