Exploring the role of structure and dynamics in the function of chymotrypsin inhibitor 2.

Increasing awareness of the possible role of internal dynamics in protein function has led to the development of new methods for experimentally characterizing protein dynamics across multiple time scales, especially using NMR spectroscopy. A few analyses of the conformational dynamics of proteins ranging from nonallosteric single domains to multidomain allosteric enzymes are now available; however, demonstrating a connection between dynamics and function remains difficult on account of the comparative lack of studies examining both changes in dynamics and changes in function in response to the same perturbations. In previous work, we characterized changes in structure and dynamics on the ps–ns time scale resulting from hydrophobic core mutations in chymotrypsin inhibitor 2 and found that there are moderate, persistent global changes in dynamics in the absence of gross structural changes (Whitley et al., Biochemistry 2008;47:8566–8576). Here, we assay those and additional mutants for inhibitory ability toward the serine proteases elastase and chymotrypsin to determine the effects of mutation on function. Results indicate that core mutation has only a subtle effect on CI2 function. Using chemical shifts, we also studied the effect of complex formation on CI2 structure and found that perturbations are greatest at the complex interface but also propagate toward CI2's hydrophobic core. The structure–dynamics–function data set completed here suggests that dynamics plays a limited role in the function of this small model system, although we do observe a correlation between nanosecond-scale reactive loop motions and inhibitory ability for mutations at one key position in the hydrophobic core.