The folding pathway of a fast-folding immunoglobulin domain revealed by single-molecule mechanical experiments

Abstract

The F-actin crosslinker filamin from Dictyostelium discoideum (ddFLN) has a rod domain consisting of six structurally similar immunoglobulin domains. When subjected to a stretching force, domain 4 unfolds at a lower force than all the other domains in the chain. Moreover, this domain shows a stable intermediate along its mechanical unfolding pathway. We have developed a mechanical single-molecule analogue to a double-jump stopped-flow experiment to investigate the folding kinetics and pathway of this domain. We show that an obligatory and productive intermediate also occurs on the folding pathway of the domain. Identical mechanical properties suggest that the unfolding and refolding intermediates are closely related. The folding process can be divided into two consecutive steps: in the first step 60 C-terminal amino acids form an intermediate at the rate of 55 s(-1); and in the second step the remaining 40 amino acids are packed on this core at the rate of 179 s(-1). This division increases the overall folding rate of this domain by a factor of ten compared with all other homologous domains of ddFLN that lack the folding intermediate.

Figure 1

Single-molecule mechanics of ddFLN1–5. (A) Structure of the ddFLN1–5 construct comprising immunoglobulin domains 1–5 including domain 4 (blue). (B) Force–extension curve of ddFLN1–5 unfolding. A total of four immunoglobulin domains are unfolded. Domain 4 unfolding (highlighted in blue) proceeds by means of a stable unfolding intermediate, whereas other immunoglobulin domains are two-state unfolders. (Nuclear magnetic resonance structures are adapted from Fucini et al (1997) and Schwaiger et al (2004).) Orange lines are worm-like chain fits using P=0.5 nm and contour lengths as indicated above the peaks (Bustamante et al, 1994).

Figure 2

Domain 4 of ddFLN has a stable refolding intermediate. (A) Mechanical refolding experiments with ddFLN4. Three typical outcomes from a series of 50 stretch and relax cycles with a single ddFLN4 domain. Black traces represent relaxation curves and red traces stretching curves. Starting from the unfolded state U (traces I, III and V), ddFLN4 can either fold back to the native state N (II), to a folding intermediate RFI (IV) or not fold at all (VI). The blue pattern superimposed in the background is a complete unfolding pattern of ddFLN4. (B) Refolding experiments from the unfolding intermediate. (I) Unfolding trace, starting from the native state N and ending in the unfolding intermediate state UFI. (II) Domain 4 is relaxed and allowed to refold. (III) Subsequent stretching shows that ddFLN4 has folded back to the native state N from UFI. This experiment is repeated in traces IV and V. (C) Kinetic scheme of the mechanical unfolding and refolding pathways of ddFLN4.

Figure 3

Double-jump mechanical single-molecule experiment. (A) Time course of the mechanical extension (upper left) and corresponding force-extension curve of a single double jump. (B) Time course of the mechanical extension for a typical experiment running through 50 unfolding–refolding cycles (upper right). Lower right: superposition of the corresponding force-extension traces. Sample traces for the three possible outcomes of the refolding experiments are coloured in blue (complete refolding), red (refolding to the intermediate RFI) and orange (no refolding).

Figure 4

Single-molecule folding and unfolding kinetics of ddFLN4. (A) Fraction of observed events for mechanical refolding as a function of the allowed refolding time (red: no refolding; green: complete refolding; blue: refolding to RFI). For each time point at 5, 10, 20, 30 and 40 ms, 1,377, 404, 417, 536 and 270 events were analysed, respectively. The lines are fits according to the kinetic folding scheme shown at the top with k₁=55±4 s⁻¹ and k₂=179±20 s⁻¹ (see Methods). (B) Histograms of unfolding forces for unfolding from native state N to UFI (left), from RFI to unfolded state U (middle) and from UFI to U (right). The solid lines in the left and middle histograms are fits according to a two-state model (see text and Methods).

Figure 5

Mechanical unfolding and refolding pathways in the energy landscape. (A) Schematic view of the ddFLN4 folding and unfolding energy landscape. The native state N and the intermediate state I correspond to minima in the energy landscape. The mechanical unfolding pathway (black line) and the folding pathway (red) are probably different. However, they both proceed through the same intermediate state. (B) Projection of the mechanical unfolding pathway on the mechanical reaction coordinate (N–C-terminal distance).