Cooperative sub-millisecond folding kinetics of apomyoglobin pH 4 intermediate.
For small single-domain proteins, formation of the native conformation (N) from a fully unfolded form (U) or from a partially folded intermediate (I) occurs typically in a highly cooperative process that can be described by a two-state model. However, it is not clear whether cooperativity arises early along the folding reaction and whether folding intermediates are also formed in highly cooperative processes. Here, we show that each previously identified step leading apomyoglobin from its unfolded form to its native form, namely, the U <= => Ia, the Ia <= => Ib, and the Ib <= => N reactions, exhibits typical features of a two-state reaction. First, refolding and unfolding kinetics of the earliest U <= => Ia reaction are measurable at pH 4.2 within the urea-induced unfolding transition [Jamin, M., and Baldwin, R. L. (1996) Nat. Struct. Biol. 3, 613-618; Jamin, M., and Baldwin, R. L. (1998) J. Mol. Biol. 276, 491-504], and we report here that sub-millisecond kinetics measured by far-UV circular dichroism (CD), a probe of secondary structure, are similar to those measured by Trp fluorescence, a probe of hydrophobic core formation and chain collapse. These results confirm that folding of the earliest intermediate, Ia, occurs in a highly cooperative process, in which hydrophobic collapse and secondary structure formation occur concomitantly in the A(B)GH core. Second, when the refolding of N is measured at high pH, starting from the acid-unfolded ensemble, the formation of Ia occurs in the mixing time of the sub-millisecond stopped-flow, but the subsequent steps, the Ia <= => Ib and Ib <= => N reactions, exhibit similar kinetics by far-UV CD and Trp fluorescence, indicating that these two late stages of the apoMb folding process also occur in highly cooperative, two-state reactions.
Weisbuch
Sebastien
S
Laboratoire de Biophysique Moléculaire et Cellulaire, Université Joseph Fourier, BMC/DRDC, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.
Gérard
Francine
F
Pasdeloup
Marielle
M
Cappadoro
Jéremy
J
Dupont
Yves
Y
Jamin
Marc
M
D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014907:Whales
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D014364:Tryptophan