Carbonyl ¹³C transverse relaxation measurements to sample protein backbone dynamics

Abstract

Carbonyl13 C relaxation experiments to study protein backbone dynamics have recently been developed. However, the effect of three-bond 13 C -13 C couplings on transverse relaxation measurements appears not to have been considered, and the potential to detect and quantify motions on the millisecond to microsecond time scale has not been fully explored. The present paper addresses these two issues. Simulations and experiments show that scalar couplings between adjacent backbone carbonyl carbon nuclei and between backbone and side-chain carbonyl/carboxyl carbon atoms in Asp and Asn residues interfere with the accurate determination of transverse relaxation rates by Carr-Purcell-Meiboom-Gill or on-resonance spin-lock measurements. The use of off-resonance radio-frequency fields avoids efficient cross-polarization, and offers a route towards accurate R 1r measurements. In addition, this approach yields dispersion in the transverse relaxation rate as a function of the effective field when conformational exchange is present. In the case of calcium-bound calbindin D 9k , 13 C off-resonance R 1r measurements yielded uniform values of R 2 along the polypeptide chain, indicating homogeneous chemical shift anisotropies and restricted dynamics on the picosecond to nanosecond time scale. Variation of R 2 as a function of the effective spin-lock field strength was not observed for any residue, indicating the absence of large-scale conformational changes of the protein backbone in the millisecond to microsecond time window. The absence of relaxation induced by internal motions on these wide-ranging time scales reinforces the view that calcium-loaded calbindin D 9k is extremely rigid. In contrast, for the C-terminal tryptic fragment of calmodulin containing the E140Q mutation we observed widespread exchange broadening. From the carbonyl transverse relaxation dispersion profile of Asp129 the exchange rate was determined to be 28 000 s −1 .

Correlated dynamics of consecutive residues reveal transient and cooperative unfolding of secondary structure in proteins

“…Dynamic processes with similar correlation times are often detected in multiple locations of the molecule, raising the question of whether the underlying motions are correlated (corresponding to concerted fluctuations involving many atoms distributed across extended regions of the molecule) or uncorrelated (corresponding to independent fluctuations involving few atoms in localized regions). Here, we have used 13 C ␣ (i ؊ 1)͞ 13 C ␣ (i) differential multiple-quantum spin relaxation to provide direct evidence for correlated dynamics of consecutive amino acid residues in the protein sequence. By monitoring overlapping pairs of residues (i ؊ 1 and i, i and i ؉ 1, etc.…”

“…Relaxation dispersion experiments have been developed that quantify conformational exchange contributions to R 2 as a function of the strength of the applied radio-frequency fields, implemented either as CPMG (Carr-Purcell-Meiboom-Gill) pulse trains or continuous-wave spin-locks (8)(9)(10). In principle, NMR spectroscopy offers the possibility to monitor conformational exchange processes at every position along the protein backbone and side chains, provided that suitable stable isotopes (e.g., 13 C and 15 N) have been incorporated (4,(11)(12)(13)(14)(15). Importantly, the chemical shifts of different nuclei are sensitive to different types of intramolecular motions, which provides a major motivation for pursuing multinuclear studies of conformational exchange (14)(15)(16)(17)(18)(19)(20)(21).…”

“…The possibility to monitor the extent of crosscorrelation between the motions of separate sites in a protein offers an important test of whether the detected dynamic modes are localized or extend across contiguous regions of the structure. Correlated conformational exchange of neighboring 13 C ␣ spin pairs is expected to be particularly powerful for addressing secondary structure fluctuations, because the 13 C ␣ chemical shift depends mainly on the local backbone -dihedral angles (36)(37)(38). Furthermore, the MQ relaxation rates are relatively sensitive to conformational exchange in the case of 13 C ␣ , because the major dipolar interactions can be greatly reduced in perdeuterated samples, and the chemical shielding anisotropy (CSA) is small.…”

“…Here, we have measured the differential MQ relaxation rates involving 13 C ␣ spins in adjacent residues of E140Q-Tr2C [the E140Q mutant of the C-terminal domain of calmodulin (Tr2C)]. We have previously demonstrated that the calcium-loaded form of E140Q-Tr2C exchanges between two major conformations that resemble the calcium-loaded (open) and calcium-free (closed) states of WT Tr2C (11,39,40).…”

“…We have previously demonstrated that the calcium-loaded form of E140Q-Tr2C exchanges between two major conformations that resemble the calcium-loaded (open) and calcium-free (closed) states of WT Tr2C (11,39,40). Based on SQ 15 N, 1 H, and 13 C ␣ rotating-frame relaxation (R 1 ) measurements, the average exchange correlation times at 28°C are ͗ ex ͘ ϭ 22 Ϯ 7, 19 Ϯ 7, and 25 Ϯ 8 s, respectively, and the populations of the exchanging states are approximately equal (11,14,15). These properties make E140Q-Tr2C a good model system for studying fast conformational exchange in general and the dynamics of the conformational switch in calmodulin in particular.…”