Home » Solution structure of a minor and transiently formed state of a T4 lysozyme mutant

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant

Bouvignies, G., Vallurupalli P., et al. Nature. 477(7362), 111-4. (2011)

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers). However, a rigorous understanding of the relation between a protein’s structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules and populates an excited state transiently (about 1 ms) to about 3% at 25 6C. We show that such binding occurs only via the ground state, and present the atomic-level model of the ‘invisible’, excited state obtained using a combined strategy of relaxation-dispersion NMR and CSRosetta model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein’s function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

bouvigines11aa-c, Selected regions from 1H-13C HSQC spectra (recorded at 1 degree C) of (a) L99A, G113A T4L (b) and L99A, G113A, R119P T4L (c), with the peaks from the ground and excited states colored in blue and red, respectively. d-f, Corresponding energy landscapes, showing the structures of the ground and excited states and their fractional populations