Carbonyl carbon transverse relaxation dispersion measurements and ms-μs timescale motion in a protein hydrogen bond network

A constant-time, Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation, R-2, dispersion experiment for carbonyl carbons was designed and executed to detect mus-ms time-scale dynamics of protein backbone carbonyl sites. Because of the large (ca. 55 Hz) C-alpha- C' J-coupling, the carbonyl signal intensity is strongly modulated as the spacing between CPMG pulses is varied, in uniformly C-13 enriched proteins, unless care is taken to minimize the perturbation of the C-alpha magnetization by the CPMG pulses. CPMG pulse trains consisting of either a band-selective pulse, such as RE-BURP, or rectangular ( with an excitation null in the C-alpha region of the spectrum) pulses were employed in order to minimize C' signal modulation by C-alpha-C' J-coupling. The performance of these types of CPMG refocusing pulses was assessed by computer simulation, and by comparing dispersion profiles measured for (1) uniformly [C-13, N-15, H-2] (H-2 at non-labile hydrogen sites) labeled, and (2) uniformly N-15/selectively-C-13 labeled samples of HIV-1 protease bound to a potent inhibitor, DMP323. In addition, because the uniformly C-13/N-15/H-2 labeled sample was well suited to measure N-15 and H-1 R-2 dispersion as well as C-13' dispersion, conformational exchange in the inter subunit beta-sheet hydrogen-bond network of the inhibitor-bound protease was elucidated using relaxation dispersion data of all three types of nuclei.