New methods and experiments in protein NMR

Jun Xu, Purdue University

Abstract

Nuclear Magnetic Resonance (NMR) is one of the principal tools of structural biology. In particular, it is exquisitely sensitive to certain fine structural features and to internal dynamics of biological macromolecules. The first part of the dissertation focuses on the backbone 15N spin relaxation experiment as a probe of internal protein dynamics. The standard suite of experiments targeting protonated amide groups, 15N[ 1H], has been augmented with a new HA(CACO)N-type scheme allowing for relaxation measurements at the deuterated sites, 15N[ 2H]. Due to the distinctive gyromagnetic ratios of the 15N and 2H spins, this new experiment is sensitive to the spin transitions at a uniquely low frequency, (ωN+ω D)/2π=26 MHz vs. ωH=500 MHz, thus facilitating studies of slower motional processes. The new technique has been demonstrated on a sample of B1 immunoglobulin binding domain from peptostreptococcal protein L. In the second part of the thesis study, a different strategy has been adopted to highlight nanosecond time scale internal dynamics. Specifically, a series of protein samples have been prepared in water/glycerol solvent with the goal to slow down the protein overall tumbling and thus to increase the separation between τR (characteristic time of rotational diffusion) and τ s (characteristic time of slow internal motion). In interpreting the data, backbone 15N relaxation rates were used to determine τ R and the obtained results were subsequently employed in the analysis of the side-chain methyl 13C relaxation rates. Using this approach, a positive identification of the nanosecond time-scale (τs=2 ns) rotameric jumps has been achieved for one of the side chains in the SH3 domain from chicken α-spectrin. Despite the widely recognized success of NMR as a tool for protein structure determination, there is no accepted measure for the accuracy of NMR structures. In the third chapter of this work, it is shown how a set of independently measured 15N- 1H residual dipolar couplings (RDC) can be used to assess the accuracy of protein coordinates. In particular, an empirical relationship is derived between the RDC figure of merit and the effective resolution of the structural model. The method is demonstrated on a sample of PDZ2 domain from human phosphatase hPTP1E. Finally, in the fourth section a uniquely sensitive structural probe, 1HN chemical shift temperature coefficients, is considered. Using this parameter, it was possible to judge the degree of agreement between the solution- and solid-state structures of the α-spectrin SH3 domain.

Degree

Ph.D.

Advisors

Skrynnikov, Purdue University.

Subject Area

Analytical chemistry

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