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 bifunctionalhydrophilic hydrophobicself assembled monolayersSAMsas a substrate that interacts with the bicelle envelope and indirectly preconcentrates and preorganizes membrane proteins to facilitate nucleation and crystal growthOf thehuman membrane proteins potentially relevant to drug responsehave yet to be structurally characterized due in large part to challenges in crystallizationWith andgtof pharmaceuticals and other drugs targeting membrane proteinsproductivity improvements in protein crystallization that enable structural characterization will have a significant impact on Public HealthObtaining diffraction quality crystals is a key bottleneck in protein crystal structure determinationFunctional enhancements to crystallization surfaces can improve nucleation and crystallization outcomes for membrane proteinsTangible benefits including a more efficient use of the proteinincreased yield of crystalline materialand reduced times to crystallization onset will improve the understanding of signalling and responses for substances of abusepharmaceutical developmentand other areas of biomedical researchThe 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 proteinThis strategy preserves membrane protein solubility and conformation and acts to enhance preorganization and improves the propensity to achieve crystal nucleationThe hypothesis is that bifunctional SAMs interacting with a bicelle envelope will indirectly preconcentrate and preorganize membrane proteins to improve crystal nucleationSpecific AimCreatebifunctional SAMs of varying H bonding surface moietye gdonor acceptorhydrophilic hydrophobic balanceisland heightor island density and probe ability to facilitate targeted interactions with membrane protein bicelle envelopesCollect replicate datanon crystallization outcomesY Nvscontrol surfaces for bacteriorhodopsin bicelles using sitting drop vapor diffusionand advance top six performing bifunctional SAMs for quantitative assessment in AimSpecific AimFor select bifunctional SAMsdemonstrate statistically significantnimprovements ofin crystallization outcomeY Nimprovement in crystallization onset timeorincrease in quantity of protein crystals generated per trial vscontrols for bacteriorhodopsinConfirm X ray diffraction qualitye gby diffraction resolutionmosaicityetcof bacteriorhodopsin crystals produced on SAM surfacesPhase II will expand studies to other high value membrane protein targets that are recalcitrant to crystallizationDeNovX 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 Project NarrativeRelevance to Public Health Research The role of structural biology in facilitating an understanding of biological function is well establishedand improvements in crystallization methods for membrane proteins are needed as this remains a key rate limiting step in the fieldAn acceleration in the acquisition of structural biology information will positively impact Public Health by expanding the structure function knowledge base for membrane proteinswhich will lead to a better understanding of drug action for both pharmaceuticals and substances of abuse