Volkman et al. employed ¹⁵N nuclear magnetic resonance (NMR) relaxation studies to characterize the fast subnanosecond and slower micro- to millisecond motions of the backbone in three forms of NtrC: the unphosphorylated (inactive) and phosphorylated (active) states and a partially activated unphosphorylated mutant. Fast backbone motion was found to be largely the same across all three states. In contrast, regions of the inactive protein that changed structure upon activation showed micro- to millisecond motions of the backbone that were suppressed upon activation. The authors concluded that these results demonstrated that activation of NtrC occurs in the micro- to millisecond range, and not in the pico- or nanosecond time regime. They further suggested that functionally important motions are likely to occur in the micro- to millisecond time regime because biological processes occur there. This is arguable from at least two viewpoints:

1) Volkman et al. discounted an allosteric role for subnanosecond motion on the basis of time scale. In fact, allosteric regulation concerns the free energy coupling between ligation, structure change, and function. It is fundamentally thermodynamic in nature (2, 3). The time scales of structural transitions and functional events need not correlate with the rates of interconversion of multiple states. Thus, although the transition from one form to another may indeed occur in a given time regime, the origins of allosteric activation may arise elsewhere, i.e., in the conformational entropy underlying the dynamics.

2) Volkman et al. further assume that the dynamic behavior of the main chain necessarily represents that of the entire protein. This conclusion is suspect. Although there are clear instances in which main dynamics report on the thermodynamics of protein function [e.g., (4)], it has become clear that fast side chain motion is also important in this context (5-7). In contrast to the backbone, side chains display a large range of dynamic angular disorder on the subnanosecond time scale, and this disorder is heterogeneously distributed. These results point to a considerable residual protein entropy (6, 7) that can potentially contribute to allosteric phenomena (8). It is important to note that no definitive correlation between main chain and side chain motion is found.

Ironically, the calmodulin system used Volkman et al. (1) to buttress the generality of their conclusions serves to illustrate the points made here. The cooperative binding of calcium to calmodulin results in the transition of the apo-state, characterized by extensive backbone motion on the micro- to millisecond time scale, to the holo-state, where these motions are largely damped (9). Volkman et al. highlight this but ignore the fact that the subsequent transition from the calcium-activated holo-state to a complex with a target domain results in no significant change in the fast dynamics of the backbone (like NtrC), but does result in a major reduction of fast side chain dynamics, corresponding to an impressive loss of ~35 kcal per mole of conformational entropy (10).

In summary, in contrast to what is asserted by Volkman et al., the time scale of functionally relevant motion is not necessarily restricted by the relevant functional rate constant, side chain dynamics cannot in general be predicted on the basis of main chain dynamics, and the changes in side chain conformational entropy, expressed in the subnanosecond time regime, should not be excluded a priori as a contributor to allosteric free energy transduction in proteins. These interesting emerging issues will have to be resolved by direct experiment in individual cases, including NtrC.

A. Joshua Wand
The Johnson Research Foundation and
Department of Biochemistry & Biophysics
University of Pennsylvania
Philadelphia, PA 19104-6059, USA
E-mail: wand{at}mail.med.upenn.edu

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Response: Wand points out that side chain motions are important for protein function and that they cannot in general be predicted on the basis of main chain dynamics. He further states that the "origins" of allosteric changes may arise on time scales faster than those of the conformational transitions themselves. These concepts are fundamental principles of protein biochemistry with which we have no disagreement, and our report (1) made no assertions regarding these principles.

We showed that (i) both the inactive and active states of the signaling protein NtrC are populated before phosphorylation, (ii) activation occurs by a shift of this preexisting equilibrium, and (iii) structural rearrangement proceeds on the microsecond time scale (1). These experimental conclusions are not in dispute here. All additional concepts and assumptions discussed by Wand were not made in our publication. It is common knowledge that there is no definite correlation between side chain and backbone dynamics. In no way did we imply that the conformational exchange between inactive and active states monitored by backbone dynamics is necessarily correlated with side chain motion. We chose to study backbone dynamics exclusively as the most appropriate marker for large conformational rearrangements of the backbone.

Further, we did not claim to unravel the origins of allosteric activation. Current understanding of the details of thermodynamic compromise that lead to the structure of globular proteins is still limited and, consequently, one is even further away from a quantitative evaluation of the individual contributions to subtle changes in conformational energy.

In summary, Wand takes us to task for conclusions that were not made in our report. Neither our results nor our conclusions are discordant with the issues discussed by Wand.

Dorothee Kern
Department of Biochemistry
Brandeis University
Waltham, MA 02454, USA
E-mail: dkern{at}brandeis.edu

REFERENCES