title: Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding.
abstract: There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.
authors.affiliation: Biomolecular Structure and Design Program, Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610, USA.
authors.forename: George W N
authors.initials: GW
authors.lastname: White
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
authors.forename: J Gunter
authors.initials: JG
authors.lastname: Grossmann
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
authors.forename: Valerie
authors.initials: V
authors.lastname: Daggett
mesh_terms: D003198:Computer Simulation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D020598:Proto-Oncogene Proteins c-myb / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014508:Urea
chemical_list: D020598:Proto-Oncogene Proteins c-myb; D014508:Urea