CARBONYL CARBON PROBE OF LOCAL MOBILITY IN C-13,N-15-ENRICHED PROTEINS USING HIGH-RESOLUTION NUCLEAR-MAGNETIC-RESONANCE

The utility of carbonyl carbons as probes of internal mobility in prot eins is investigated by theoretical and experimental methods. In a dou ble C-13,N-15-labeled sample, the relaxation of the carbonyl carbon is mediated by dipolar interactions with nearby protons, the C-13(alpha) and N-15 nuclei, and the C-13 chemical shielding anisotropy (CSA). Ex pressions are presented for carbonyl single-spin, carbonyl-nitrogen, a nd carbonyl-alpha-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 derive d, because the CSA autocorrelation dominates carbonyl relaxation. Prot on-detected C-13,N-15 NMR spectroscopy is used to measure one-spin car bonyl and two-spin carbonyl-nitrogen relaxation rates. Measurements ar e performed at 9.4, 11.7, and 17.6 T for carbonyl carbons in villin 14 T, the N-terminal 14 kDa domain of the actin-binding protein villin. T hree rate measurements are used to obtain the values of the spectral d ensity function at zero [J(0)], nitrogen [J(omega(N))], and carbonyl [ J(omega(C))] frequencies. The different secondary structural elements such as alpha-helices, beta-sheets, and regions of low persistent stru cture have distinctive dynamic behavior that the values of the spectra l density function at low frequencies (<75 MHz) reveal. The value of J (0) is especially sensitive to both rapid and slow internal motions an d is discussed in detail. Comparison with N-15-only data indicates tha t one can obtain similar dynamic information from the carbonyl data. I n addition, carbonyl NMR studies are potentially useful for probing hy drogen-bond dynamics, as significantly different average J(0) values w ere observed for hydrogen-bonded and solvent-exposed carbonyls.