A novel NMR experiment allows one to characterize slow motion in macromolecules. The method exploits the fact that motions, such as rotation about dihedral angles, induce correlated fluctuations of the isotropic chemical shifts of the nuclei in the vicinity. The relaxation of two-spin coherences involving Cα and Cβ nuclei in proteins provides information about correlated fluctuations of the isotropic chemical shifts of Cα and Cβ. The difference between the relaxation rates of double- and zero-quantum coherences and is shown to be affected by cross-correlated chemical shift modulation. In ubiquitin, evidence for slow motion is found in loops or near the ends of β-strands and α-helices.
The fundamental advantages of using pulsed field gradients to improve multidimensional NMR experiments are now well established. Unwanted resonances, such as that of water in spectra of biomolecules, can easily be eliminated ( I ) , and coherences belonging to groups of nuclei of particular interest can be efficiently selected (2). Many experiments also inherently provide quadrature detection in the indirectly detected dimension because of the selection of a single coherence pathway (+2wr -P ~2) as opposed to a linear combination of pathways that results in amplitude modulation of a single quadrature channel (3). Less frequently addressed are problems associated with maintaining optimum resolution of cross peaks in 2D plots (4). Straightforward implementation of quadrature detection in the indirect dimension often produces phase-twisted peaks. This results because of the direct correlation of phases of coherence in one dimension with phases of magnetization in the other dimension (phase modulation of signal by t, evolution). For non-gradient-enhanced versions of 2D and 3D experiments these problems have traditionally been addressed using hypercomplex data acquisition in a manner such as that described by States et al. (5)) or time-proportional phase incrementation in a manner such as that described by Marion and Wtithrich (6). Neither of these procedures is directly applicable to gradient-enhanced spectroscopy because of selection of a single coherence pathway. Here we take a lead from a recent paper by Hurd et al. ( 7) that employs changes in the sign of gradient pulses to retrieve both positive and negative coherence pathways in a single experiment. The procedure described in that paper places high demands on gradient pulse length and may not, at this stage of hardware development, be generally applicable. However, a procedure using gradient changes in alternate t, points provides a simple alternative. Here, we acquire hypercomplex data for a heteronuclear multiple-quantum-coherence (HMQC) experiment. The data show pure amplitude modulation in both channels and transform to give pure-absorption signals. Moderate efficiency of acquisition is retained as illustrated with a 13C‘H natural-abundance HMQC data set on the trisaccharide Lewisx glucal (galactosyl-& ( 1,4) [ CX( 1,3 )-fucosyl] ghtcal). These data required just 36 min on 10 mg of the trisaccharide. Suppression of 12C lines is complete with just two pulses per t, point.
The interpretation of residual dipolar couplings in terms of molecular properties of interest is complicated because of difficulties in separating structural and dynamic effects as well as the need to estimate alignment tensor parameters a priori. An approach is introduced here that allows many of these difficulties to be circumvented when data are acquired in multiple alignment media. The method allows the simultaneous extraction of both structural and dynamic information directly from the residual dipolar coupling data, in favorable cases even in the complete absence of prior structural knowledge. Application to the protein ubiquitin indicates greater amplitudes of internal motion than expected from traditional 15N spin relaxation analysis.
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We report the de novo determination of 15N-1H bond orientations and motional order parameters for the protein ubiquitin with high accuracy based solely on NMR residual dipolar coupling measurements made in six distinct alignment media. The resulting bond orientations are in agreement with RDC-refined orientations of either solid or solution state coordinates to within approximately 2 degrees , which is also the estimated precision of the resulting orientations. The squared generalized order parameters, which reflect amplitudes of motion spanning the picosecond to millisecond time scales, exhibit a correlation with picosecond time scale order parameters derived from conventional NMR 15N spin relaxation methods. Provided that RDC measurements can be obtained using many different alignment media, this approach (called direct interpretation of dipolar couplings) may significantly impact the attainable accuracy and the molecular weight range accessible to NMR structure determination in the solution state, as well as provide a route for the study of motions occurring on the nanosecond to microsecond time scales, which have been traditionally difficult to study at atomic resolution.
