title: Solution structure of a minor and transiently formed state of a T4 lysozyme mutant.
abstract: 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 °C (ref. 5). 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 (ref. 6) and CS-Rosetta 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.
authors.affiliation: Department of Molecular Genetics, The University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bouvignies
authors.forename: Pramodh
authors.initials: P
authors.lastname: Vallurupalli
authors.forename: D Flemming
authors.initials: DF
authors.lastname: Hansen
authors.forename: Bruno E
authors.initials: BE
authors.lastname: Correia
authors.forename: Oliver
authors.initials: O
authors.lastname: Lange
authors.forename: Alaji
authors.initials: A
authors.lastname: Bah
authors.forename: Robert M
authors.initials: RM
authors.lastname: Vernon
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Dahlquist
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics*; D019143:Evolution, Molecular; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D011485:Protein Binding; D013696:Temperature
chemical_list: D008024:Ligands; D009113:Muramidase