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. 2006 Nov 28;103(48):18113-8.
doi: 10.1073/pnas.0604580103. Epub 2006 Nov 15.

Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains

Sarah Batey  1 , Jane Clarke
Affiliations

Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains

Sarah Batey et al. Proc Natl Acad Sci U S A. .

Abstract

Approximately 75% of eukaryotic proteins contain more than one so-called independently folding domain. However, there have been relatively few systematic studies to investigate the effect of interdomain interactions on protein stability and fewer still on folding kinetics. We present the folding of pairs of three-helix bundle spectrin domains as a paradigm to indicate how complex such an analysis can be. Equilibrium studies show an increase in denaturant concentration required to unfold the domains with only a single unfolding transition; however, in some cases, this is not accompanied by the increase in m value, which would be expected if the protein is a truly cooperative, all-or-none system. We analyze the complex kinetics of spectrin domain pairs, both wild-type and carefully selected mutants. By comparing these pairs, we are able to demonstrate that equilibrium data alone are insufficient to describe the folding of multidomain proteins and to quantify the effects that one domain can have on its neighbor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure of the two-domain spectrin fragment R1516. The C helix of R15 forms a continuous helix with the A helix of R16 (Protein Data Bank ID 1U5P).
Fig. 2.
Fig. 2.
Equilibrium and kinetic data for R1516. (a) Equilibrium denaturation curves of spectrin R1516 (purple), R1617 (black), and their constituent domains, R15 (blue), R16 (red), and R17 (cyan). These data follow the change in fluorescence at 350 nm (filled circles) and change in CD signal at 222 nm (open circles), which all overlay well. Note that the apparent m values of all proteins are the same, within error, as judged by the slope of the transition, except for R1617, which has an increased m value (Table 1). Data for individual domains were taken from ref. ; data for R1617 were taken from ref. . (b) A plot of the natural logarithm of the observed rate constants for R1516. Data from fluorescence (filled symbols) and CD (open symbols) measurements are shown. Closed squares show rate constants which could only be observed in double-jump, interrupted refolding experiments. Blue, data for the R15 domain in R1516 (with R16 unfolded); Red, data for the R16 domain in R1516 (with R15 folded). For details on the assignment of the phases, see Supporting Text. (Note that a third, proline isomerization-limited phase was also observed in refolding experiments, but this has been omitted for clarity; see Supporting Text).
Fig. 3.
Fig. 3.
Kinetics of R1617 wild-type and mutants. The data shown in red represent the folding and unfolding of the R16 domain in R1617, and the data shown in cyan represent the folding and unfolding data for the R17 domain in R1617. The data shown in filled symbols were determined by using single-jump stopped flow measurements. The data shown in open circles could only be observed in double-jump, interrupted unfolding experiments. (a) The kinetics of R1617 wild-type (data taken from ref. where details of the full assignment of the kinetic phases can also be found). (b) The kinetics of R1617 S20A. As for wild-type at [urea] below ≈6 M, only a single unfolding phase can be detected. (c) The kinetics of R1617 L203A. Two unfolding phases are observed at all [urea]. Note that the rollover in the folding kinetics of R1617 reflects dead-time formation of a collapsed intermediate, which is marginally stable, has little secondary structure, and is not populated at equilibrium (6).
Fig. 4.
Fig. 4.
The folding pathways of R1516 (a) and R1617 (b). The rate constants shown are the folding and unfolding rates extrapolated to 0 M denaturant. In both cases, the N-terminal domain folds first, followed by the C-terminal domain. In R1617, there is also a low stability partly folded early intermediate (I1) that has little secondary structure but can be detected by a dead-time change in fluorescence (6).
Fig. 5.
Fig. 5.
Chevron plots for R15 and R16 alone and in R1516. The dependence of the observed rate constants for folding and unfolding against [urea]. Blue filled circles, R15 domain alone; blue open circles, R15 in R1516; the solid lines are fits to a two-state equation. In R1516, R15 is folding and unfolding in the presence of an unfolded R16 domain. The folding rate constants are the same as for R15 alone, but R15 unfolds more slowly in R1516. Red filled circles, R16 domain alone; red open circles, R16 in R1516; the solid lines are fits to a sequential transition state model (41, 42). In R1516, R16 is folding and unfolding in the presence of a folded R15 domain. The R16 domain folds more rapidly and unfolds more slowly in R1516.
Fig. 6.
Fig. 6.
The equilibrium populations of the native (N), intermediate (I), and denatured (D) species at different denaturant concentrations (determined from the kinetic rate constants). (a) R1516. (b) R1617 wild-type. (e) R1617 with the mutation S20A. (d) R1617 with the mutation L203A. The filled circles represent the population of N, the open circles represent the population of I (folded R15 in R1516 and folded R16 in R1617 wild type and mutants), and the filled triangles represent the population of D. The population of I is negligible at all denaturant concentrations for wild-type R1617 and the apparently cooperative mutant S20A. I is significantly populated in R1516 and the “noncooperative” mutant of R1617, L203A. These data were used to model the expected equilibrium data for R1516 (c) and wild-type R1617 (d). It was assumed that the two domains had the same CD signal (as they have approximately the same number of helical residues) and that R16 (two Trp residues) has double the fluorescence change of R15 or R17 (one Trp each). The modeled and experimental data overlay. Thus, it is apparent that it is the population of an intermediate at equilibrium that leads to a lower equilibrium m value in R1516 (and the L203A mutant of R1617) than in wild-type R1617.
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