Over the last ten years, single-molecule force spectroscopy has proven to be extremely useful in studying the unfoldingenergy landscapes of proteins. One major advantage of this new approach is the precise control of the reaction coordinate. In earlier force spectroscopy experiments, the reaction coordinate was mainly constrained to the N–C-terminal direction of the protein. However, recently the toolkit to design pulling geometries along almost arbitrary force directions was extended by disulfide engineering of polyproteins. In those experiments, a strong anisotropy of the unfolding-energy landscape was observed. Unfolding rates varying by several orders of magnitude were found along the various pulling directions. To date, the effects of force on the folding pathway have only been rarely studied, owing to the much lower forces involved in active refolding and the associated technical demands. Herein, we describe the design of single-molecule experiments to study the anisotropy of the folding mechanics of a protein under external force. The idea and experimental realization of our experiment is depicted in Figure 1. The conventional geometry for studying the mechanics of protein folding is shown in the scenario at the top (blue). A polypeptide chain is held at its N and C termini, and hence the mechanical force will act on the whole chain while the protein is folding. To study the effect of force on protein folding, it would be desirable to compare the N–C-terminal pulling geometry with other geometries in which the mechanical force only acts on part of the chain (middle and bottom scenarios in Figure 1). We used the protein ubiquitin, which has been characterized in unfolding and refolding experiments. Recently, it was shown that ubiquitin folds against mechanical loads applied in the N–Cterminal direction. To realize the three pulling geometries of Figure 1, we used cysteine engineering, which allowed us to change the sites of force application. The force was applied through residues 1 and 76 in the first pulling geometry (blue), 1 and 35 in the second geometry (red), and 1 and 16 in the third geometry (green). The parts of the polypeptide chains exposed to force during folding are colored in the three protein structures shown in Figure 1. Attachment of the N terminus of ubiquitin to the cantilever tip and the surface occurred through three immunoglobulin (Ig) domains of human titin (I91–I93) fused to the N terminus of ubiquitin. On average, the titin domains unfold at higher forces, while refolding occurs with kinetics one to two orders of magnitude slower than for ubiquitin. The cysteine residues introduced into ubiquitin at positions 76, 35, or 16 ensured dimerization, resulting in constructs as shown in the rightmost column of Figure 1. The unfolding fingerprint of the two ubiquitin domains sandwiched between Ig domains was clearly observable in unfolding traces (see the Supporting Information). We used the following protocol for mechanical refolding experiments: First, both ubiquitin domains and one to three Ig-handle domains were unfolded. Afterwards, the unfolded, stretched polypeptide chain was relaxed with a continuous velocity vp= 5 nms !1 down to an extension of approximately 20 nm above the surface. Subsequently, the polypeptide chain was stretched again with the same pulling velocity back to the starting extension. To minimize drift artifacts in the force– extension traces and to increase the force resolution, we performed these experiments with a lock-in detection adding a small oscillation amplitude of 7 nm on the tip movement, as described by Schlierf et al. (see also the Supporting Information). This additional lock-in signal can be used in cases for which instrumental drift complicates identification of refolding events. Those folding events can be identified by clear, discrete events in the lock-in traces (see reference [7] and the Supporting Information). Figure 2a–c shows typical force–extension folding traces (colored) and subsequent unfolding traces (gray) for the three different ubiquitin constructs Ubi1,76, Ubi1,35, and Ubi1,16. All traces exhibit two Figure 1. Anisotropy of folding mechanics under force. The conventional design of force experiments between the N and C termini is illustrated in the top scenario (blue). Different pulling directions result in a partly constrained polypeptide chain during active folding and are shown in the middle and bottom scenarios (red and green). The protein ubiquitin allowed the experimental realization with the three shown constructs. The attachment to the surface and the cantilever was achieved through Ig-handles (gray triangles).