Advanced Nucleation Technologies for Membrane Protein Crystallization to Accelerate Structure-Based Drug Design for Substance Use Disorders - 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.
