It is proposed to obtain effective Lipari-Szabo order parameters and local correlation times for relaxation vectors of protein (13)CO nuclei by carrying out a (13)CO-R(1) auto relaxation experiment, a transverse (13)CO CSA/13CO-13Calpha CSA/dipolar cross correlation and a transverse (13)CO CSA/(13)CO-(15)N CSA/dipolar cross correlation experiment. Given the global rotational correlation time from (15)N relaxation experiments, a new program COMFORD (CO-Modelfree Fitting Of Relaxation Data) is presented to fit the (13)CO data to an effective order parameter S2CO, an effective local correlation time and the orientation of the CSA tensor with respect to the molecular frame. It is shown that the effective S2CO is least sensitive to rotational fluctuations about an imaginary Calpha-Calpha axis and most sensitive to rotational fluctuations about an imaginary axis parallel to the NH bond direction. As such, the Calpha-Calpha information is fully complementary to the (15)N relaxation order parameter, which is least sensitive to fluctuations about the NH axis and most sensitive to fluctuations about the Calpha-Calpha axis. The new paradigm is applied on data of Ca(2+) saturated Calmodulin, and on available literature data for Ubiquitin. Our data indicate that the S2CO order parameters rapport on slower, and sometimes different, motions than the (15)N relaxation order parameters. The CO local correlation times correlate well with the calmodulin's secondary structure.
Residue-specific amide proton spin-flip rates K were measured for peptide-free and peptide-bound calmodulin. K approximates the sum of NOE build-up rates between the amide proton and all other protons. This work outlines the theory of multi-proton relaxation, cross relaxation and cross correlation, and how to approximate it with a simple model based on a variable number of equidistant protons. This model is used to extract the sums of K-rates from the experimental data. Error in K is estimated using bootstrap methodology. We define a parameter Q as the ratio of experimental K-rates to theoretical K-rates, where the theoretical K-rates are computed from atomic coordinates. Q is 1 in the case of no local motion, but decreases to values as low as 0.5 with increasing domination of sidechain protons of the same residue to the amide proton flips. This establishes Q as a monotonous measure of local dynamics of the proton network surrounding the amide protons. The method is applied to the study of proton dynamics in Ca(2+)-saturated calmodulin, both free in solution and bound to smMLCK peptide. The mean Q is 0.81 +/- 0.02 for free calmodulin and 0.88 +/- 0.02 for peptide-bound calmodulin. This novel methodology thus reveals the presence of significant interproton disorder in this protein, while the increase in Q indicates rigidification of the proton network upon peptide binding, confirming the known high entropic cost of this process.
NMR spin relaxation experiments provide a powerful tool for the measurement of global and local biomolecular rotational dynamics at subnanosecond time scales. Technical limitations restrict most spin relaxation studies to biomolecules weighing less than 10 kDa, considerably smaller than the average protein molecular weight of 30 kDa. In particular, experiments measuring z , the
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