X-ray compatible microfluidic platforms for studying protein structure and function

If you can look into the seeds of time, And say which grain will grow and which will not. - Macbeth. Act I, Scene 3. Knowledge of the three dimensional structure of a protein provides insight into its mechanism, as well as into potential sites for drug targeting. Often these insights provide functional information about an unknown protein, lead to a deeper understanding of how damaged proteins cause disease, and potentially reveal a path to new drug treatments. The most common method to determine protein structure and function is to crystallize them and probe them with X-rays to determine their structure. Protein crystallization still remains an art rather than an exact science, and it is extremely difficult to predict the conditions that will ultimately result in crystals resulting in difficulties in structure determination. Recent efforts in structural biology have helped to lessen bottlenecks using robotics and/or microfluidics, leading to improved, and often automated, high throughput methodologies for screening, crystallization, and X-ray diffraction analysis of novel protein targets. Even with these improvements, the harvesting and mounting of protein crystals for X-ray analysis remains a cumbersome, manual step in the structural biology pipeline that especially hampers collection of data from small/fragile crystals. Obtaining high-quality protein crystals is the main obstacle for structure elucidation because crystallization is a complex, multi-parametric process that involves setting up thousands of crystallization trials to screen a vast chemical space. Current data collection strategies involve harvesting a single crystal from the crystallization droplet in which it was grown, a process that often damages the crystal, followed by cryocooling the crystal, before subjecting it to monochromatic X-rays to elucidate the structure from X-ray data. For de novo structure determination, phase information also needs to be collected to convert diffraction data (that records intensities) to structural information. Phase information is most commonly obtained experimentally by measuring anomalous diffraction signal. The collection of high quality phasing data is dependent on having diffraction data with a high signal-to-noise ratio while minimizing radiation-induced damage and crystal non-isomorphism. The microfluidic crystallization platform developed in this work serves to overcome issues with current methods in protein structure determination. Specifically, it allows for screening (crystallization) conditions together with on-chip X-ray analysis of crystals formed, and can be further used for relevant functional studies of proteins via time resolved crystallography. Furthermore, this is the first example of a microfluidic chip being used for room temperature phasing from diffraction data collected and merged from multiple crystals. Microfluidics offers more precise control over the composition and the kinetics of a crystallization trial than what is possible using conventional methods such as vapor diffusion and microbatch. The ability to control fluid flow and transport at such a small preparative scale makes it an ideal platform for crystallization since microfluidic devices use minimal amounts of protein solution, which are often times not available in large quantities for medically relevant human targets. The use of appropriate polymers in chip fabrication enables on-chip analysis thus eliminating the need for crystal harvesting prior to analysis. Identification of these materials and development of protocols to fabricate X-ray compatible microfluidic platforms is discussed in Chapter 2. Proof-of-principle studies using model systems that validate the microfluidic chip and an alternate data collection strategy for merging datasets from multiple crystals is covered in Chapter 3. Screening of conditions and ¬de novo structure determination via on-chip collection of phase information is discussed in further detail in Chapter 4. In Chapter 5, the application of microfluidic platforms for use in seeding studies is discussed. Seeding is a method to improve chances of obtaining high quality crystals by separating the nucleation and growth phases in a crystallization trial, and this chapter focusses on how it was achieved using a microfluidic platform. Laue crystallography allows us to look at protein molecules in motion, helping us understand the complex pathways and structural changes that accompany protein function. Chapter 6 describes how X-ray transparent microfluidic chips can be used for collecting time resolved Laue data thus enabling functional studies on protein targets that would be cumbersome using traditional methods. The platform developed here can also be used to enable structural studies that shed light on the change in structure during protein function, ranging from proteins that respond to stimuli such as light, pH, or the presence of ligands, to those that respond to temperature changes, thus allowing an unprecedented opportunity for a range of previously elusive biological studies