title: Recognition between disordered states: kinetics of the self-assembly of thioredoxin fragments.
abstract: The disordered N- (1-73) and C- (74-108) fragments of oxidized Escherichia colithioredoxin (Trx) reconstitute the native structure upon association [Tasayco, M. L., & Chao, K. (1995) Proteins: Struct., Funct., Genet. 22, 41-44]. Kinetic measurements of the formation of the complex (1-73/74-108) at 20 degrees C under apparent pseudo-first-order conditions using stopped-flow far-UV CD and fluorescence spectroscopies indicate association coupled to folding, an apparent rate constant of association [kon = (1330 +/- 54) M-1 s-1], and two apparent unimolecular rate constants [k1 = (0. 037 +/- 0.007) s-1 and k2 = (0.0020 +/- 0.0005) s-1]. The refolding kinetics of the GuHCl denatured Trx shows the same two slowest rate constants. An excess of N- over C-fragment decreases the kon, and the slowest phase disappears when a P76A variant is used. Stopped-flow fluorescence measurements at 20 degrees C indicate a GuHCl-dependent biphasic dissociation/unfolding process of the complex, where the slowest phase corresponds to 90% of the total. Their rate constants, extrapolated to zero denaturant, k-1 = (9 +/- 3) x 10(-5) s-1 and k-2 = (3.4 +/- 1.2) x 10(-5) s-1, show m# values of (4.0 +/- 0.4) kcal mol-1 M-1 and (3.5 +/- 0.1) kcal mol-1 M-1, respectively. Our results indicate that: (i) a compact intermediate with trans P76 and defined tertiary structure seems to participate in both the folding and unfolding processes; (ii) not all the N-fragment is competent to associate with the C-fragment; (iii) conversion to an association competent form occurs apparently on the time scale of P76 isomerization; and (iv) the P76A variation does not alter the association competency of the C-fragment, but it permits its association with "noncompetent" forms of the N-fragment.
authors.affiliation: Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
authors.forename: A F
authors.initials: AF
authors.lastname: Chaffotte
authors.forename: J H
authors.initials: JH
authors.lastname: Li
authors.forename: R E
authors.initials: RE
authors.lastname: Georgescu
authors.forename: M E
authors.initials: ME
authors.lastname: Goldberg
authors.forename: M L
authors.initials: ML
authors.lastname: Tasayco
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry*; D015870:Gene Expression; D019791:Guanidine; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D013879:Thioredoxins; D019791:Guanidine