Temperature Dependence of the Internal Dynamics of a Calmodulin−Peptide Complex

Abstract

The temperature dependence of the fast internal dynamics of calcium-saturated calmodulin in complex with a peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light chain kinase is examined using 15N and 2H NMR relaxation methods. NMR relaxation studies of the complex were carried out at 13 temperatures that span 288-346 K. The dynamics of the backbone and over four dozen methyl-bearing side chains, distributed throughout the calmodulin molecule, were probed. The side chains show a much more variable and often considerably larger response to temperature than the backbone. A significant variation in the temperature dependence of the amplitude of motion of individual side chains is seen. The amplitude of motion of some side chains is essentially temperature-independent while many show a simple roughly linear temperature dependence. In a few cases, angular order increases with temperature, which is interpreted as arising from interactions with neighboring residues. In addition, a number of side chains display a nonlinear temperature dependence. The significance of these and other results is illuminated by several simple interpretative models. Importantly, analysis of these models indicates that changes in generalized order parameters can be robustly related to corresponding changes in residual entropy. A simple cluster model that incorporates features of cooperative or conditional motion reproduces many of the unusual features of the experimentally observed temperature dependence and illustrates that side chain interactions result in a dynamically changing environment that significantly influences the motion of internal side chains. This model also suggests that the intrinsic entropy of interacting clusters of side chains is only modestly reduced from that of independent side chain motion. Finally, estimates of protein heat capacity support the view that the major contribution to the heat capacity of protein solutions largely arises from local bond vibrations and solvent interactions and not from torsional oscillations of side chains.

Entropy redistribution controls allostery in a metalloregulatory protein

“…3C), because an inverse temperature dependence can be indicative of conditional motion, attributed to excluded volume effects (39) and possibly other correlated motional processes (39,47,48). Most of the residues in the apo-and Zn 2 -bound CzrA homodimers are well described by a harmonic or mildly anharmonic potential (0 < Λ < 3; Fig.…”

“…3B), we further probed the origin of these dynamical changes. The increase in flexibility likely derives from the release of restrictions imposed on rotamer populations by neighboring residues, associated with conditional motion (39,40). Such motions have also been described as dynamical networks and are more frequently interrogated by molecular dynamics simulations from crystal structures (17,(41)(42)(43)(44), combined, in some cases, with NMR fast timescale dynamics analysis (17,45,46).…”

Dynamic personalities of proteins

“…Because these fast-timescale dynamics are, ultimately, connected to entropy, NMR spectroscopy has been used extensively to investigate the entropic contribution of the protein to biomolecular binding (in protein-protein, protein-DNA, protein-RNA and protein-other-ligand interactions) [65][66][67][68][69] . Here, we use calmodulin to illustrate the advantages and limitations of this method for quantifying entropic contributions to affinity, because the natural function of calmodulin is to bind to a variety of target proteins.…”

“…5a). However, the conversion of order parameters into absolute entropic energies is challenging and controversial [65][66][67][68][69]72,73 . A trend correlating the entropy calculated from the methyl-NMR order parameters of calmodulin and the total binding entropy of the system obtained from isothermal titration calorimetry is observed 72 (Fig.…”

Characterization of the Fast Dynamics of Protein Amino Acid Side Chains Using NMR Relaxation in Solution

“…92,93 For the simple models discussed here, the parametric relationship between the measurable generalized order parameter and the corresponding residual entropy are easily calculated ( Figure 15). 92,93 In this context, the results of Lee et al 106 are particularly interesting when viewed from the point of view of the underlying thermodynamics of binding of target domains by calmodulin. Wintrode & Privalov 113 attempted to dissect the change in conformational entropy of calmodulin upon binding the smMLCKp domain from the system thermodynamic parameters obtained from calorimetric measurements.…”