title: Side-chain interactions in the folding pathway of a Fyn SH3 domain mutant studied by relaxation dispersion NMR spectroscopy.
abstract: A major challenge to the study of protein folding is the fact that intermediate states along the reaction pathway are generally unstable and thus difficult to observe. Recently developed NMR relaxation dispersion experiments present an avenue to accessing such states, providing kinetic, thermodynamic, and structural information for intermediates with small (greater than or equal to approximately 1%) populations at equilibrium. We have employed these techniques to study the three-state folding reaction of the G48M Fyn SH3 domain. Using (13)C-, (1)H-, and (15)N-based methods, we have characterized backbone and side-chain interactions in the folded, unfolded, intermediate, and transition states, thereby mapping the energy landscape of the protein. We find that the intermediate, populated to approximately 1%, contains nativelike structure in a central beta-sheet, and is disordered at the amino and carboxy termini. The intermediate is stabilized by side-chain van der Waals contacts, yet (13)C chemical shifts indicate that methyl-containing residues remain disordered. This state has a partially structured backbone and a collapsed yet mobile hydrophobic core and thus closely resembles a molten globule. Nonpolar side-chain contacts are formed in the unfolded-intermediate transition state; these interactions are disrupted in the intermediate-folded transition state, possibly allowing side chains to rearrange as they adopt the native packing configuration. This work illustrates the power of novel relaxation dispersion experiments in characterizing excited states that are "invisible" in even the most sensitive of NMR experiments.
authors.affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Anthony
authors.initials: A
authors.lastname: Mittermaier
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Korzhnev
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D000818:Animals; D002645:Chickens; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009154:Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics*
chemical_list: C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn