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

Columbia University, New York NY

Investigators

Abstract

In this project the PI will develop new solid state NMR methods to study protein structure. In the previous grant period, the PI has largely completed the site-specific NMR resonance assignments for microcrystalline human Ubiquitin, providing proof of principle for the first stage in denovo structure determination by solid state NMR. In this project, the PI will confirm the existing assignments, and locate signals for the mobile portions. With the recent availability of high magnetic field strengths, her group intends to attempt more challenging and biologically interesting targets; two additional proteins would be assigned as solid phase precipitates: Calmodulin and Triosephosphate Isomerase. Finally, the PI will determine the 3D structure of Ubiquitin based on solid-state NMR data, utilizing several sources of experimental data. Torsional constraints are available from the chemical shifts themselves, which will be analyzed using statistical databases from solution NMR as well as from computed shifts. Hydrogen bond correlations will be measured via N-H...C correlations. Tertiary contacts will be measured using spin diffusion as well as selective recoupling experiments. Structures would be calculated using commercially available software. While structural genomics proceeds apace, many important non-soluble proteins are not readily characterized; the PI's laboratory will attempt characterization of some of the non-soluble targets when the methods are established through this work. The PI is involved with collaborations with several academic and industrial labs that will facilitate transfer of the information obtained through this research. The core effort, however, is the training of outstanding graduate students. High school and undergraduate students will be trained and exposed to structural biology and magnetic resonance in the PI's laboratory.