# Characterization of protein motions in the intermediate timescale via MAS NMR

> **NIH NIH F32** · COLUMBIA UNIV NEW YORK MORNINGSIDE · 2020 · $67,446

## Abstract

Abstract: The rotating frame relaxation (R1ρ) experiment is a powerful probe of microsecond to
millisecond timescale motions, and has been developed and applied for solution NMR studies of
biopolymers. Recent applications of rotating frame relaxation in magic angle spinning solid-state
NMR studies highlight an analogous, possibly more powerful opportunity to study dynamics of
biopolymers. However, if anisotropic interactions are large, confounding coherent evolution
processes make it difficult to accurately determine the exchange effects. While amide 15N
measurements have been performed and interpreted, sites with larger anisotropic interactions
such as the carbonyl or aromatic groups with their large chemical shift anisotropy (CSA) have
been impossible. We recently developed a pulse sequence that nearly eliminates coherent
evolution during the spin lock, while the exchange effects can still be observed (called “refocused
CSA rotating frame relaxation” or RECRR). We plan to characterize the ability of the RECRR
experiment to analyze 13C carbonyl sites, focusing on slow intermediate exchange motions (k < ~
103 s-1). Coherent contributions due to dipolar couplings during the RECRR experiment appear to
be small, but will be characterized in detail using numerical simulations and experimental data
using phenylalanine•HCl as a model system, and with this information in mind we will develop
isotopic enrichment protocols for these experiments. We will perform relaxation dispersion
measurements on the protein ubiquitin, contrasting RECRR and the traditional spin lock, and
characterizing the ability of these two experiments to obtain quantitative descriptions of the
motion. Since RECRR is able to probe both 13C carbonyls and 15N amide sites, this experiment
should uncover significantly more insights into the mechanism of motion for protein backbone
sites. Motion models derived from simulations of these data will be evaluated using subsequent
data at additional field strengths. Finally, we plan to apply these methods to study activation and
inactivation of the potassium ion channel, KcsA. We contrast the dynamics of the Activated state
in exchange with the Deactivated state, vs. the Activated state in exchange with the Deactivated
state, both of which are expected to involve slow to intermediate motions. Previous work suggests
that clusters of amino acids are associated with the allosteric coupling between activation and
inactivation process. The proposed dynamics measurements will clarify the molecular processes
involved.

## Key facts

- **NIH application ID:** 10008964
- **Project number:** 5F32GM135350-02
- **Recipient organization:** COLUMBIA UNIV NEW YORK MORNINGSIDE
- **Principal Investigator:** Eric George Keeler
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $67,446
- **Award type:** 5
- **Project period:** 2019-08-01 → 2021-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10008964

## Citation

> US National Institutes of Health, RePORTER application 10008964, Characterization of protein motions in the intermediate timescale via MAS NMR (5F32GM135350-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10008964. Licensed CC0.

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