Temperature Dependence of Anisotropic Protein Backbone Dynamics

Abstract

The measurement of (15)N NMR spin relaxation, which reports the (15)N-(1)H vector reorientational dynamics, is a widely used experimental method to assess the motion of the protein backbone. Here, we investigate whether the (15)N-(1)H vector motions are representative of the overall backbone motions, by analyzing the temperature dependence of the (15)N-(1)H and (13)CO-(13)C(alpha) reorientational dynamics for the small proteins binase and ubiquitin. The latter dynamics were measured using NMR cross-correlated relaxation experiments. The data show that, on average, the (15)N-(1)H order parameters decrease only by 2.5% between 5 and 30 degrees C. In contrast, the (13)CO-(13)C(alpha) order parameters decrease by 10% over the same temperature trajectory. This strongly indicates that there are polypeptide-backbone motions activated at room temperature that are not sensed by the (15)N-(1)H vector. Our findings are at variance with the common crank-shaft model for protein backbone dynamics, which predicts the opposite behavior. This study suggests that investigation of the (15)N relaxation alone would lead to underestimation of the dynamics of the protein backbone and the entropy contained therein.

Determination of Chemical Shift Anisotropy Tensors of Carbonyl Nuclei in Proteins through Cross‐Correlated Relaxation in NMR

“…At this point, there is no conclusive evidence that would allow one to assess the exact nature of anisotropic internal motions in ubiquitin. [30,42,43] To evaluate the importance of the assumption of isotropic internal motions, we Figure 1 that specifies the orientation of the CSA tensors with respect to the environment. As explained in the text, the nth peptide plane was assumed to be characterized by a single order parameter S 2 n = S 2 ( 15 N n + 1 ), and were taken from the work of Tjandra et al [41] The angles q ii,C'X , required to calculate the rates of Equation (2) were taken from the NMR structure.…”

Local Structure and Anisotropic Backbone Dynamics from Cross‐Correlated NMR Relaxation in Proteins

“…This important result demonstrates that the different relaxation rates provide a consistent description of the local anisotropic dynamics. This observation also argues that out-of-plane motion of the NÀH bond vector [9,32] does not have a strong effect on these relaxation rates.…”

“…Recently, the vector fluctuations of N À H and C'ÀC a bonds were monitored by studying the temperature dependence of cross-correlated relaxation rates rather than their absolute values, thereby diminishing the influence of uncertainties in CSA parameters. [9] With extended DFT calculations, we previously found significant site-specific variations in both the magnitude of the C' CSA (the relative magnitudes of the three principal components of the shift tensor d 11 , d 22 , and d 33 ) and the orientation of the C' chemical-shift tensor (with principal axes x 11 , x 22 , and x 33 ; Figure 1). We formulated a simple model for the C' CSA, which depends only on the isotropic chemical shift, d iso .…”

“…[1][2][3][4] In this respect, CCR rates involving CSA tensors are most useful, as they sample local structure and dynamics along three orthogonal axes. Over the past few years, numerous NMR experiments have been designed to measure crosscorrelated relaxation rates to determine local molecular geometry, [3,5] to study local dynamics, [6][7][8][9] and to characterize chemical-shift tensors in solution.[10-12] Different models of anisotropic local motion of the peptide plane have been discussed. [7,9,[13][14][15] Whereas CCR processes in principle contain an enormous amount of information, the interpretation of these relaxation rates must be treated with great care.…”

“…Over the past few years, numerous NMR experiments have been designed to measure crosscorrelated relaxation rates to determine local molecular geometry, [3,5] to study local dynamics, [6][7][8][9] and to characterize chemical-shift tensors in solution.…”