Many biological processes generate force, and proteins have evolved to resist and respond to tension along different force axes. Single-molecule force spectroscopy allows for molecular insight into the behavior of proteins under force and the mechanism of protein folding in general. Here, we have used src SH3 to investigate the effect of different pulling axes under the low-force regime afforded by an optical trap. We find that this small cooperatively folded protein shows an anisotropic response to force; the protein is more mechanically resistant to force applied along a longitudinal axis compared to force applied perpendicular to the terminal β strand. In the longitudinal axis, we observe an unusual biphasic behavior revealing a force-induced switch in the unfolding mechanism suggesting the existence of two parallel unfolding pathways. A sitespecific variant can selectively affect one of these pathways. Thus, even this simple two-state protein demonstrates a complex mechanical unfolding trajectory, accessing multiple unfolding pathways under the low-force regime of the optical trap; the specific unfolding pathway depends on the perturbation axis and the applied force. Many cellular processes such as protein folding/unfolding, protein degradation, and nucleic acid splicing are mechanical processes in which force plays a significant physiological role (1, 2). The advent of single-molecule force spectroscopy has allowed the observation of these events at an unprecedented resolution and yielded significant mechanistic details (3, 4). This single-molecule force spectroscopy approach has also provided insight into our basic understanding of how proteins fold and unfold (5-7). In mechanical unfolding experiments, the applied force privileges a particular direction in space along which a welldefined reaction coordinate-namely, the end-to-end extension of the molecule-emerges as a natural metric of the extent of the reaction (1,8,9). The location of the transition state along this defined reaction coordinate can be determined directly from such experiments. This information, in combination with recent theoretical approaches (10), can provide detailed information on the protein folding free energy landscape (11).Several studies have investigated the factors influencing the mechanical response of proteins under force; most of these studies are under the high-force regime of the atomic force microscopy (AFM). The term "mechanical stability" refers to a protein's average unfolding force at a given loading rate. Because most of these AFM studies are carried out under high loading rates, they are far from equilibrium; the mechanical stability reflects the kinetic likelihood to unfold and is therefore not a measure of stability in the thermodynamic sense. These experimental studies have led to the suggestion that native-state topology governs mechanical stability (2, 12, 13). In general, β-sheet proteins appear to be mechanically stronger than α-helical proteins (12). The network of hydrogen bonds between adjacent β strands ...

A fundamental question in protein folding is whether proteins fold through one or multiple trajectories. While most experiments indicate a single pathway, simulations suggest proteins can fold through many parallel pathways. Here, we use a combination of chemical denaturant, mechanical force and site-directed mutations to demonstrate the presence of multiple unfolding pathways in a simple, two-state folding protein. We show that these multiple pathways have structurally different transition states, and that seemingly small changes in protein sequence and environment can strongly modulate the flux between the pathways. These results suggest that in vivo, the crowded cellular environment could strongly influence the mechanisms of protein folding and unfolding. Our study resolves the apparent dichotomy between experimental and theoretical studies, and highlights the advantage of using a multipronged approach to reveal the complexities of a protein's free-energy landscape.

The recent revolution in optics and instrumentation has enabled the study of protein folding using extremely low mechanical forces as the denaturant. This exciting development has led to the observation of the protein folding process at single molecule resolution and its response to mechanical force. Here, we describe the principles and experimental details of force spectroscopy on proteins, with a focus on the optical tweezers instrument. Several recent results will be discussed to highlight the importance of this technique in addressing a variety of questions in the protein folding field.