Abstract
We have used NMR and computational methods to characterize the dynamics of the ribonuclease barnase over a wide range of timescales in free and inhibitor-bound states. Using temperature- and denaturant-dependent measurements of chemical shift, we show that barnase undergoes frequent and highly populated hinge bending. Using relaxation dispersion, we characterize a slower and less populated motion with a rate of 750 ± 200 s−1, involving residues around the lip of the active site, which occurs in both free and bound states and therefore suggests conformational selection. Normal mode calculations characterize correlated hinge bending motions on a very rapid timescale. These three measurements are combined with previous measurements and molecular dynamics calculations on barnase to characterize its dynamic landscape on timescales from picoseconds to milliseconds and length scales from 0.1 to 2.5 nm. We show that barnase has two different large-scale fluctuations: one on a timescale of 10−9−10−6 s that has no free energy barrier and is a hinge bending that is determined by the architecture of the protein; and one on a timescale of milliseconds (i.e., 750 s−1) that has a significant free energy barrier and starts from a partially hinge-bent conformation. These two motions can be described as hierarchical, in that the more highly populated faster motion provides a platform for the slower (less probable) motion. The implications are discussed. The use of temperature and denaturant is suggested as a simple and general way to characterize motions on the intermediate ns-μs timescale.
Highlights
It is generally accepted that the dynamic landscape of a protein is critical for its function (Hammes-Schiffer and Benkovic, 2006)
Curvature of temperature dependence provides an unreliable guide to the location of alternative states, because the amount of curvature is affected both by the chemical shift difference between the two states, and by the free energy difference between the is very close to zero, and there is no significant dependence of c on [guanidinium hydrochloride (Gdm)]
For 16 residues, a well-defined dependence could be observed. The shapes of these dependences vary: in general, a concave shape shows that the excited state has a chemical shift greater than the ground state whereas a convex shape shows that the excited state has a smaller chemical shift
Summary
It is generally accepted that the dynamic landscape of a protein (that is, the set of conformations it can adopt, and the rates at which it can move between them) is critical for its function (Hammes-Schiffer and Benkovic, 2006). It is important to recognize that even the slower transitions still involve atomic movement on a rapid timescale; the more highly correlated motions just occur infrequently and with low probability, and for that reason can be called “slower”. Such motions are difficult to characterize experimentally, but can be described using molecular dynamics and other computational techniques (Gao et al, 2006; Ma and Nussinov, 2010; Ramanathan and Agarwal, 2011). An examination of the lowest energy normal modes shows that, in agreement with the expectation above, they generally involve movements of rigid domains around small hinge regions
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