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

The utility of carbonyl carbons as probes of internal mobility in proteins is investigated by theoretical and experimental methods. In a double 13 C, 15 N-labeled sample, the relaxation of the carbonyl carbon is mediated by dipolar interactions with nearby protons, the 13 C α and 15 N nuclei, and the 13 C 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 13 C, 15 N 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 (ω N )], and carbonyl [ J (ω C )] 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 15 N-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.