Research Project
9615631
PROJECT SUMMARY The goal of this Phase I proposal is to improve membrane protein crystallization outcomes using bifunctional (hydrophilic/hydrophobic) self-assembled monolayers (SAMs) as a substrate that interacts with the bicelle envelope and indirectly preconcentrates and preorganizes membrane proteins to facilitate nucleation and crystal growth. Of the ? 4700 human membrane proteins potentially relevant to drug response, 94% have yet to be structurally characterized due in large part to challenges in crystallization. With > 60% of pharmaceuticals and other drugs targeting membrane proteins, productivity improvements in protein crystallization that enable structural characterization will have a significant impact on Public Health. Obtaining diffraction quality crystals is a key bottleneck in protein crystal structure determination. Functional enhancements to crystallization surfaces can improve nucleation and crystallization outcomes for membrane proteins. Tangible benefits including a more efficient use of the protein, increased yield of crystalline material, and reduced times to crystallization onset will improve the understanding of signalling and responses for substances of abuse, pharmaceutical development, and other areas of biomedical research. The typical SAM will constitute a patterned bifunctional monolayer of chemically tunable end-groups that indirectly facilitates preconcentration and preorganization of the membrane protein through interactions with the bicelle envelope surrounding the membrane protein. This strategy preserves membrane protein solubility and conformation and acts to enhance preorganization and improves the propensity to achieve crystal nucleation. The hypothesis is that bifunctional SAMs interacting with a bicelle envelope will indirectly preconcentrate and preorganize membrane proteins to improve crystal nucleation. Specific Aim 1 - Create ? 20 bifunctional SAMs of varying H-bonding surface moiety (e.g., donor/acceptor), hydrophilic/hydrophobic balance, island height, or island density and probe ability to facilitate targeted interactions with membrane protein bicelle envelopes. Collect replicate data (n ? 6) on crystallization outcomes (Y/N) vs. control surfaces for bacteriorhodopsin bicelles using sitting drop vapor diffusion, and advance top six performing bifunctional SAMs for quantitative assessment in Aim 2. Specific Aim 2 - For select bifunctional SAMs, demonstrate statistically significant (n ? 12) improvements of ? 7% in crystallization outcome (Y/N), ? 15% improvement in crystallization onset time, or ? 30% increase in quantity of protein crystals generated per trial vs. controls for bacteriorhodopsin. Confirm X-ray diffraction quality (e.g., by diffraction resolution, mosaicity, etc.) of bacteriorhodopsin crystals produced on SAM surfaces. Phase II will expand studies to other high value membrane protein targets that are recalcitrant to crystallization. DeNovX?s innovative approaches to nucleation of membrane proteins will expand the structure/function understanding in drug response and accelerate structure-based design of new targets.
