Carbonyl Carbon Probe of Local Mobility in¹³C,¹⁵N-Enriched Proteins Using High-Resolution Nuclear Magnetic Resonance

Abstract

The utility of carbonyl carbons as probes of internal mobility in proteins is investigated by theoretical and experimental methods. In a double 13C,15N-labeled sample, the relaxation of the carbonyl carbon is mediated by dipolar interactions with nearby protons, the 13Cα and 15N nuclei, and the 13C chemical shielding anisotropy (CSA). Expressions are presented for carbonyl single-spin, carbonyl-nitrogen, and carbonyl-α-carbon two-spin rates due to dipolar interaction and a CSA tensor. We show that, at high magnetic fields, useful relations between relaxation rates and spectral density functions can be derived, because the CSA autocorrelation dominates carbonyl relaxation. Proton-detected 13C,15N NMR spectroscopy is used to measure one-spin carbonyl and two-spin carbonyl-nitrogen relaxation rates. Measurements are performed at 9.4, 11.7, and 17.6 T for carbonyl carbons in villin 14T, the N-terminal 14 kDa domain of the actin-binding protein villin. Three rate measurements are used to obtain the values of the spectral density function at zero [J(0)], nitrogen [J(ωN)], and carbonyl [J(ωC)] frequencies. The different secondary structural elements such as α-helices, β-sheets, and regions of low persistent structure have distinctive dynamic behavior that the values of the spectral density function at low frequencies (<75 MHz) reveal. The value of J(0) is especially sensitive to both rapid and slow internal motions and is discussed in detail. Comparison with 15N-only data indicates that one can obtain similar dynamic information from the carbonyl data. In addition, carbonyl NMR studies are potentially useful for probing hydrogen-bond dynamics, as significantly different average J(0) values were observed for hydrogen-bonded and solvent-exposed carbonyls.