National Biomedical Resource for
Advanced ESR Spectroscopy

New Methods for Analyzing Heteronuclear NMR Relaxation Data from Proteins

Proteins can be uniformly isotope-labeled with ¹⁵N, ¹³C and ²H using recombinant DNA techniques. The capability of measuring and interpreting the spin relaxation properties of these heteronuclei turns NMR into a valuable method which can provide a complete dynamic map of the protein. The amide ¹⁵N nucleus is a particularly useful probe, relaxed primarily by dipolar coupling to the amide proton and by ¹⁵N chemical shift anisotropy (CSA). Therefore ¹⁵N relaxation is controlled by the dynamic processes experienced by the N-H bond vector, which includes overall rotational reorientation and local motions. Thus far, the spin relaxation data have been interpreted with the simplified model-free approach which assumes complete dynamic decoupling between overall and internal motions in proteins. In order to gain physically meaningful insight into protein dynamics, it is necessary to use theoretical models that account for coupling among dynamic modes. We developed a new and novel comprehensive theoretical methodology that does just that. It consists of an adaptation to NMR of the ESR version of the slowly relaxing local structure (SRLS) model of Freed and co-workers. The basic tenets of ¹⁵N NMR and spin probe ESR relaxation are very similar. Within the scope of SRLS the faster motion is used to describe the internal dynamics of the N-H moiety, while the slower motion accounts for the global rotation of the protein. A computer program that calculates spectral densities was used to generate a grid of spectral densities. This program generates theoretical T₁, T₂ and NOE data. In the first successful application, we studied the enzyme Adenylate kinase from E. coli in its ligand-free (AK) and AP₅A-bound (AKC) form with ¹⁵N spin relaxation. It catalyzes the reaction AMP+ATP↔2ADP. The figure on the left shows regions of high (colored blue) and low (colored red) ordering and the one on the right shows fast (blue) and slow (red) motions. We were able to show that loops α₄/β₃ and α₅/β₄ in AKC engage in dynamics that are related to the “energetic counter-balancing of substrate binding” effect that apparently drives the kinase catalysis. Other flexible loops in AKC may relate to domain motion toward product release.

Publication: V. Tugarinov, Y.E. Shapiro, Z. Liang, J.H. Freed, and E. Meirovitch, J. Mol. Biol., 315, 155-170 (2002); no PMCID

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ACERT is supported by grant 1R24GM146107 from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.