The increasing output of new integral membrane protein (IMP) structures is partially due to the availability of an increasing number of chemical variables such as detergents, detergent-like molecules, lipids, and solubilizing/stabilizing additives. It is clear that specific combinations and concentrations of these different variables are often necessary to improve IMP solubility/stability and crystallization. For each IMP studied, researchers typically explore (via a trial and error approach) many of these chemical variables. However, there is limited understanding of the importance and effect of specific combinations/concentrations of these chemical variables on IMP extraction, solubilization/stabilization, purification and crystallization. We propose to use our novel technology and approach, high-throughput self-interaction chromatography (HSC) combined with an incomplete factorial screen and artificial neural network analysis to discover new formulations that improve/optimize IMP solubility and physical stability (IMP homogeneity). This phase-1 proposal will perform a pilot study on seven different IMPs including the chemokine receptor-1 (CCR1), cystic fibrosis transmembrane regulator protein (CCR1), epithelial sodium channel (ENaC), acid sensing ion channel (ASIC), P- glycoprotein (Pgp), bacteriorhodopsin (BR) and ferric yersiniabactin uptake receptor (FIP). The HSC approach involves rapid experimental determination of second viral coefficient values (B- values) for an incomplete factorial of the total possible number of chemical combinations and concentrations for each IMP. This is followed by input and analysis via an artificial neural network (ANN) program that, once "trained", produces B-value predictions for a complete factorial of possible chemical combinations and concentrations. Chemical conditions associated with the most positive B-value predictions (suggesting improved solubility and physical stability) are then experimentally validated via HSC measurements followed by evaluation of the IMP's stability/folding (via differential scanning calorimetry, Western gels, CD and when available, bio- assays), solubility and stability in several of these solutions. It is possible that the proposed comprehensive analysis of chemical variables will result in new combinations and/or concentrations that may become useful "cocktails" to be included in screening solubility and stability new IMPs within the same class of IMPs. However, the major goal of the proposed research is to demonstrate the value of the HSC technology and approach to determine novel and improved solubilization/stabilization solutions.
Public Health Relevance Statement: Public Health Relevance: Researchers typically explore a variety of detergents and detergent/lipid combinations in attempts to enable isolation, purification of integral membrane proteins (IMPs) to support biological, drug discovery and structural biology studies. IMPs, important proteins involved in human disease, are the targets of more than 50% of all drugs currently under development. The inability to express, solubilize and purify homogeneous preparations represents a major bottleneck in IMP research adversely affecting drug development pipeline. This proposal involves use of a novel technology and experimental approach to determine novel combinations and concentrations of existing detergents and additives that can improve the solubility, stability and crystallization of IMPs.
Project Terms: Affect; ASIC channel; Bacteriorhodopsins; Biological; Biological Assay; Biological Neural Networks; Caliber; Chemicals; chemokine receptor; Chromatography; Crystallization; Cystic Fibrosis Transmembrane Conductance Regulator; Detergents; Development; Differential Scanning Calorimetry; drug development; drug discovery; Drug Formulations; Eligibility Determination; ENaC (epithelial Na+ channel); Epithelial; Evaluation; Excipients; Gel; Goals; human disease; improved; Integral Membrane Protein; Lipids; Measurement; Membrane Proteins; new technology; novel; novel strategies; Output; P-Glycoprotein; Pathway Analysis; Pharmaceutical Preparations; Phase; Pilot Projects; Preparation; programs; protein structure; Proteins; public health relevance; receptor; Research; Research Personnel; screening; Sodium Channel; Solubility; Solutions; structural biology; System; Technology; Training; uptake; Viral; yersiniabactin
