title: Folding dynamics of the B1 domain of protein G explored by ultrarapid mixing.
abstract: For many proteins, compact conformations are known to accumulate in advance of the rate-limiting step in folding. To understand the nature and significance of these early conformational events, we employed ultrarapid mixing methods to fully characterize the kinetics of folding of the 57-residue B1 domain of protein G. Continuous-flow fluorescence measurements exhibit a major exponential phase on the submillisecond time scale (600-700 micros), which is followed by a slower phase with a denaturant-dependent time constant (2-30 ms) observable by conventional stopped-flow measurements. The combined kinetic traces quantitatively account for the total change in Trp 43 fluorescence upon folding, including the previously unresolved 'burst phase' signal. The denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state mechanism, U right harpoon over left harpoon I right harpoon over left harpoon N, where I is a productive intermediate with native-like fluorescence properties. The relatively slow rate and exponential time course of the initial folding phase indicates that a substantial free energy barrier is encountered during chain condensation, resulting in a partially organized ensemble of states distinct from the initial unfolded conformations.
authors.affiliation: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: Park
authors.forename: M C
authors.initials: MC
authors.lastname: Shastry
authors.forename: H
authors.initials: H
authors.lastname: Roder
mesh_terms: D005453:Fluorescence; D019791:Guanidine; D007700:Kinetics; D008956:Models, Chemical; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D012997:Solvents; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000378:metabolism
chemical_list: C029152:G-substrate; D009419:Nerve Tissue Proteins; D012997:Solvents; D014364:Tryptophan; D019791:Guanidine