15N nuclear magnetic resonance relaxation studies on rat beta-parvalbumin and the pentacarboxylate variants, S55D and G98D.
15N relaxation data for Ca(2+)-bound rat beta-parvalbumin (a.k.a. oncomodulin) were analyzed using the Lipari-Szabo formalism and compared with existing data for rat alpha-parvalbumin. Although the average S(2) values for the two proteins are very similar (0.85 for alpha, 0.84 for beta), residue-by-residue inspection reveals systematic differences. alpha tends to have the lower S(2) value in helical regions; beta tends to have the lower value in the loop regions. Rat beta was also examined in the Ca(2+)-free state. The 59 assigned residues displayed an average order parameter (0.90) significantly greater than the corresponding residues in the Ca(2+)-loaded form. The pentacarboxylate variants of rat beta-S55D and G98D-also were examined in the Ca(2+)-bound state. Although both mutations significantly heighten Ca(2+) affinity, they utilize distinct energetic strategies. S55D improves the Ca(2+)-binding enthalpy; G98D improves the binding entropy. They also show disparate peptide backbone dynamics. Whereas beta G98D displays an average order parameter (0.87) slightly greater than that of the wild-type protein, beta S55D displays an average order parameter (0.82) slightly lower than wild-type beta. Furthermore, whereas just two backbone N-H bonds in beta G98D show internal motion on the 20-200-psec timescale, fully 52 of the 93 residues analyzed in beta S55D show this behavior. These findings suggest that the increased electrostatic repulsion attendant to introduction of an additional carboxylate into the CD site ligand array impedes backbone vibrational motion throughout the molecule.[1]References
- 15N nuclear magnetic resonance relaxation studies on rat beta-parvalbumin and the pentacarboxylate variants, S55D and G98D. Henzl, M.T., Wycoff, W.G., Larson, J.D., Likos, J.J. Protein Sci. (2002) [Pubmed]
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