Recognition between Disordered States:  Kinetics of the Self-Assembly of Thioredoxin Fragments

• Published in: Biochemistry (Easton) 1997-12, Vol.36 (51), p.16040-16048
• Main Authors: Chaffotte, Alain F, Li, Jian-Hua, Georgescu, Roxana E, Goldberg, Michel E, Tasayco, María Luisa
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - metabolism; Circular Dichroism; Escherichia coli - chemistry; Gene Expression; Guanidine; Kinetics; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments - metabolism; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Spectrometry, Fluorescence; Thioredoxins - chemistry; Thioredoxins - metabolism; Ultracentrifugation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9708500

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 °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 °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) × 10-5 s-1 and k -2 = (3.4 ± 1.2) × 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.