SRLS Analysis of 15N-1H NMR Relaxation from the Protein S100A1: Dynamic Structure, Calcium Binding, and Related Changes in Conformational Entropy

We report on amide (N-H) NMR relaxation from the protein S100A1 analyzed with the slowly relaxing local structure (SRLS) approach. S100A1 comprises two calcium-binding EF-hands (helix-loop-helix motifs) connected by a linker. The dynamic structure of this protein, in both calcium-free and calcium-bound form, is described as the restricted local N-H motion coupled to isotropic protein tumbling. The restrictions are given by a rhombic potential, u (similar to 10 kT), the local motion by a diffusion tensor with rate constant D-2 (similar to 10(9) s(-1)), and principal axis tilted from the N-H bond at angle beta (10-20 degrees). This parameter combination provides a physically insightful picture of the dynamic structure of S100A1 from the N-H bond perspective. Calcium binding primarily affects the C-terminal EF-hand, among others slowing down the motion of helices III and IV approximately 10-fold. Overall, it brings about significant changes in the shape of the local potential, u, and the orientation of the local diffusion axis, beta. Conformational entropy derived from u makes an unfavorable entropic contribution to the free energy of calcium binding estimated at 8.6 +/- 0.5 kJ/mol. The N-terminal EF-hand undergoes moderate changes. These findings provide new insights into the calcium-binding process. The same data were analyzed previously with the extended model-free (EMF) method, which is a simple limit of SRLS. In that interpretation, the protein tumbles anisotropically. Locally, calcium binding increases ordering in the loops of S100A1 and conformational exchange (R-ex) in the helices of its N-terminal EF-hand. These are very unusual features. We show that they most likely stem from problematic data-fitting, oversimplifications inherent in EMF, and experimental imperfections. R-ex is shown to be mainly a fit parameter. By reanalyzing the experimental data with SRLS, which is largely free of these deficiencies, we obtain-as delineated above-physically-relevant structural, kinetic, geometric, and binding information.

Automated summary

The study analyzed NMR relaxation from the protein S100A1 using the SRLS approach and found that calcium binding significantly alters the dynamic structure of the protein, providing new insights into the calcium-binding process. The findings show physically relevant structural, kinetic, geometric, and binding information free from deficiencies found in previous analyses using the EMF method.