Determination of 13C? relaxation times in uniformly 13C/15N-enriched proteins

Abstract

Relaxation times of (13)C(α) carbons of uniformly (13)C/(15)N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T(1), T(2) and the heteronuclear NOE of (13)C(α) in uniformly (13)C/(15)N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T(1) and the heteronuclear NOE were similar to those of the corresponding (15)N experiments published previously. The determination of T(2) for the C(α)-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T(2) evolution period owing to the scalar coupling of C(α)-C(β) must be suppressed. Various different pulse sequences for the T(2) evolution period were simulated in order to optimize the bandwidth for which reliable T(2) relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual (1)H-(13)C(α) cross peaks, in a series of ((1)H, (13)C)-ct-HSQC spectra with a modified CPMG sequence as well as a T(1p) sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.

THE USE OF2H,13C,15N MULTIDIMENSIONAL NMR GTO STUDY THE STRUCTURE AND DYNAMICS OF PROTEINS

“…While pulse sequences that minimize this effect in the case of methyl groups have been described (65) they do not eliminate the problem completely. Third, in the case of applications involving uniformly 13 C labeled samples both scalar and dipolar 13 C-13 C couplings must be taken into account (25,172). Although this problem is eliminated through the use of molecules where only a select number of sites are labeled the information content available in such systems is, of course, much less.…”

NMR Relaxation Mechanisms for Backbone Carbonyl Carbons in a13C,15N-Labeled Protein

“…[2] [7] where m 0 is the permeability of vacuum, ប is Plank's constant divided by 2p, g S and g I are the magnetogyric ratios of spin S and I, respectively, and r IS is the distance between spin I where s and s ⊥ are the chemical shifts of the parallel and the perpendicular directions of the direction-dependent and S. The angular Larmor frequencies of spins I and S are labeled v I and v S , respectively. chemical-shift tensor, respectively, and w is the angle be- a and 13 C, all carbon pulses were set to 64 and 54 ms pulse lengths at 11.7 and 14.1 T, respectively.…”

“…More recently also a-carbon ( 13 C a ) relaxation. Experiments designed to suppress cross correlation be-relaxation has been used to extract information about tween CSA relaxation and dipole-dipole (DD) relaxation due to backbone motions ( 6,7 ) . Additional complications asso- for which physical and geometrical parameters are uncertain, comIn this case, the 13 C magnetization would have been ex-…”

Study of protein dynamics in solution by measurement of 13C?-13CO NOE and 13CO longitudinal relaxation

“…The study of 13CO relaxation complements well-documented 15N-based relaxation measurements of the protein backbone (Nirmala and Wagner, 1988;Kay et al, 1989;Clore et al, 1990;Peng and Wagner, 1992) and ~3C~ relaxation measurements (Yamazaki et al, 1994;Engelke and Riiterjans, 1995) because additional sites are probed, the internuclear vectors involved point in different directions, and other time scales can be investigated. Homonuclear carbon-carbon cross relaxation is interesting in particular for larger proteins at larger magnetic fields, as the effect is very sensitive to differences in apparent diffusional motion at the different sites.…”