Assignment and Structural Characterization of Uniformly Labeled Proteins by Solid State NMR

Columbia University, New York NY

Investigators

Abstract

McDermott MCB 9983581 The objective of this project is to develop solid state NMR methods to characterize uniformly 13C, 15N labeled and selectively 2H labeled proteins using ubiquitin, BPTI, and margatoxin as model systems. The goal is to establish reliable assignment methods to study uncharacterized membrane systems. High resolution, magic angle spinning techniques will be emphasized in the assignment protocol in order to allow confirmation of sidechain identity through isotropic carbon shifts, in analogy to solution NMR. It has been recently demonstrated that high-resolution multidimensional solid state NMR methods can be used to correlate backbone and sidechain 13C and 15N chemical shifts of hydrated micro-crystalline U-13C and 15N BPTI. Methods for assignment will be optimized and developed for long range distance constraints and for dynamical constraints that are consistent with uniform labeling. The goal of this research is to further develop solid state nuclear magnetic resonance methods to study large and complex biological molecules. The present collection of characterized molecules is incomplete, and its particular blind spot is the fascinating proteins that rest at the perimeter of the cell, in its grease-containing membrane; here they supervise the communication and traffic between cells. Membrane proteins constitute 1/3 of the proteins of a cell, and yet only a handful of them are characterized.