CAREER: Multidimensional High Field Solid-State NMR Methods for Protein Structure Determination

University Of Illinois At Urbana-Champaign, Urbana IL

Investigators

Abstract

In this CAREER project, the PI will develop high-field magic-angle spinning solid-state NMR (SSNMR) methods and apply them to understand fundamental aspects of protein structure and dynamics. In the last few years, the first complete SSNMR structures of small peptides and proteins using uniformly-13C,15N enriched protein samples have been obtained. The PI aims to make these methods more generally applicable and improve the efficiency and quality of structure determination. The intellectual merit of this project centers on novel SSNMR methodology: (1) new chemical shift assignment experiments and protocols, employing 3D and reduced dimensionality data sampling schemes, will be established; (2) pulse sequences for measuring site-resolved chemical shift anisotropy parameters will be developed, and the experimental values compared to ab initio calculations to serve as constraints of backbone and side-chain conformation; (3) side-chain conformational exchange will be examined by analysis of chemical shift and dipolar spectra over a range of temperatures; and (4) pKa's of ionizable residues in solid proteins will be directly determined by measuring side-chain chemical shifts as a function of pH in 3D spectra. These experiments will provide several types of data that are currently unavailable to the biophysics community; for example, the data will assist in the interpretation of solution NMR relaxation experiments and directly test molecular dynamics and electrostatics calculations in solid proteins. Furthermore, the methods developed will be directly applicable to non-soluble proteins, such as membrane proteins and peptide aggregates and fibrils. Additional broader impacts of this project include educating a new generation of excellent researchers for SSNMR. Both graduate students and undergraduate researchers (drawn from liberal arts colleges and under-represented groups) will be trained in structural biology and NMR. A three-day SSNMR summer school will be held at the host institution each year during the grant period. The workshop will consist of seminars, demonstrations of modern SSNMR experiments on 500 and 750 MHz spectrometers, facility tours, and hands-on NMR software training. A dozen participants each year will be drawn from academic and industrial laboratories, with priority given to faculty from liberal arts colleges and smaller research universities. The PI will assist these educators in identifying cost-effective strategies for upgrading solution NMR spectrometers to perform magic-angle spinning experiments, and as such work to expose a larger cross-section of the scientific community to modern magic-angle spinning SSNMR.