Recognition between disordered states: Kinetics of the self-assembly of thioredoxin fragments

The disordered N-(1-73) and C-(74-108) fragments of oxidized Escherichia coli thioredoxin (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 [k(on) = (1330 +/- 54) M-1 s(-1)], and two apparent unimolecular rate constants [k(1) (0.037 +/- 0.007) s(-1) and k(2) = (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 k(on), 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.