Integral Membrane Proteins (IMPs) include many biomedically-important gate keepers, receptors,transporters, homeostasis regulators, and potential drug discovery targets. Three-dimensional (3D)structure determination of IMPs by X-ray crystallography, cryo-electron microscopy (cryo-EM), orNuclear Magnetic Resonance (NMR) methods remains a major challenge for structural biology.While NMR can generally provide accurate 3D structures of small soluble proteins, structuredetermination by solution NMR of IMPs, prepared in stabilizing membrane-mimicking environmentswhich generally require 2H,13C,15N-enrichment of the IMP, can be quite challenging. Evolutionarycouplings (ECs), evolutionarily-correlated mutations derived from multiple sequence alignments, canalso be used to provide information about native residue pair contacts, and to model the 3Dstructures of IMPs. Combining EC and NMR data provides a powerful approach for overcomingincompleteness of NMR NOESY data obtained for perdeuterated IMP samples, and the challenges inidentifying true native protein structure contacts from the phylogenetic EC analysis. In particular,inter-helical contact information that is difficult to obtain for perdeuterated IMPs by NMR is wellrepresented in the sequence co-variance EC data. Our goals are to develop a robust, reproducible,and fully automated EC-NMR platform suitable for accurate and reliable structure determination ofIMPs, particularly α-helical IMPs, and apply these methods for 3D structure analysis of biomedically-important IMPs. EC-NMR will be further developed using β-barrel and α-helical IMPs of knownstructure, and then applied to studies of IMPs of unknown structure selected from designated NIHNIAID priority pathogenic bacteria. We will (i) further develop and apply the Single Protein Production(SPP) method for producing isotope-enriched IMPs in E. coli, (ii) implement a micro-scale NMRscreening pipeline for IMP sample optimization, (iiii) rigorously and comprehensively address thequestion of how EC and NOESY data quality and quantity correlate with the accuracy of EC-NMRstructures, (iv) design improved algorithms for structure determination of IMPs combining ECs andNMR data, and (v) develop tools for validation of IMP structures determined by both conventionalNMR and EC-NMR methods. Advanced molecular modeling methods will be implemented to improveaccuracy of EC-NMR structures. ECs will also be combined with NMR data to identify and determinestructures of multiple “native states” of proteins. This study will expand the range of proteins that canbe studied by NMR, provide more accurate structural and dynamic information than can be obtainedwith existing methods, and provide fundamental structural information needed for future antibioticdrug development targeted to high-priority pathogens.
|Effective start/end date||9/1/17 → 6/30/21|
- National Institutes of Health (NIH)
Nuclear magnetic resonance
X ray crystallography