Evidence for a broad transition-state ensemble in calmodulin folding from single-molecule force spectroscopy.

In recent years, single-molecule force spectroscopy has provided a wealth of insights into protein folding and unfolding. Using mechanical force as a denaturant offers distinct advantages over thermal or chemical denaturation, such as the possibility to locally probe the folding free-energy landscape or to actively steer the unfolding pathway of proteins. However, it has been argued that the unfolding pathways that are probed in single-molecule force spectroscopy are likely to be very different from the pathways studied by chemical denaturation, which would limit or exclude any comparison between single-molecule force spectroscopy experiments and conventional folding techniques. To date, this hypothesis has only been investigated under non-equilibrium conditions, that is, under conditions that favor either unfolding or folding. With the advent of low-drift single-molecule force spectroscopy by AFM, it has recently become possible to study folding and unfolding under equilibrium conditions, thus allowing observation of multiple folding/unfolding transitions of single protein molecules in a single trace. 11] Herein, we investigate folding and unfolding pathways of the calcium signaling protein calmodulin (CaM) close to thermodynamic equilibrium by applying force in different pulling geometries. CaM is a small protein composed of two domains, both of which show two-state folding and unfolding in AFM experiments. The experiments shown herein allow us to distinguish whether folding and unfolding pathways in different pulling geometries converge onto a common pathway close to equilibrium and to investigate whether a structurally distinct zero-force pathway exists. Single-molecule force spectroscopy experiments are usually performed with protein constructs as shown in Figure 1a, where the protein of interest (i.e., CaM) is inserted on the DNA level between protein domains that serve as handles for the attachment to surface and AFM cantilever tip so that a single multi-domain protein construct is expressed. This technique is however limited to N–C-terminal pulling geometries. We have developed a method to create well-defined heteropolymers with nearly arbitrary points of force application by assembly on the protein level, which was inspired by solid-phase peptide synthesis (Figure 1b). Handle proteins with a 6 His tag are allowed to bind to a Ni-NTA resin (NTA = nitrilotriacetic acid); the protein of interest and chain-terminating handle proteins (both without His tag) are then sequentially attached by disulfide bridge formation. The cysteine residues of the protein of interest can be protected by the coupling reagent dithiodipyridine to avoid formation of homopolymers (see the Supporting Information). We have used this technique to study the unfolding kinetics of single CaM molecules in three different non-N–Cterminal directions (Figure 1c). By mutating amino acid residues 38 and 118 into cysteines (CaM 38 118), the mean unfolding forces of the Nand C-terminal domain of CaM (DomN and DomC) were increased to about 25 pN at a pulling velocity of 50 nm s , as compared to circa 13 pN in the N–C-terminal direction (Figure 1d). The measured length Figure 1. a) The experimental setup with protein construct composed of CaM (purple) and handle domains (gray). b) Stepwise synthesis of well-defined heteropolymers with alternative pulling directions. c) Structure of CaM with a sketch of the pulling geometries studied. d) Sample traces of CaM in four different pulling directions at vpull = 50 nm s . The curves have two peaks, indicating that both domains of CaM unfold independently from each other. Varying the force application point leads to significantly different unfolding forces.