Quantifying Lipari–Szabo modelfree parameters from 13CO NMR relaxation experiments

Abstract

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.