Michael Schlierf

Cysteine engineering of polyproteins for single- molecule force spectroscopy

Single-molecule methods such as force spectroscopy give experimental access to the mechanical properties of protein molecules. So far, owing to the limitations of recombinant construction of polyproteins, experimental access has been limited to mostly the N-to-C terminal direction of force application. This protocol gives a fast and simple alternative to current recombinant strategies for preparing polyproteins. We describe in detail the method to construct polyproteins with precisely controlled linkage topologies, based on the pairwise introduction of cysteines into protein structure and subsequent polymerization in solution. Stretching such constructed polyproteins in an atomic force microscope allows mechanical force application to a single protein structure via two precisely controlled amino acid positions in the functional three-dimensional protein structure. The capability for site-directed force application can provide valuable information about both protein structure and directional protein mechanics. This protocol should be applicable to almost any protein that can be point mutated. Given correct setup of all necessary reagents, this protocol can be accomplished in fewer than 10 d.

Insight into helicase mechanism and function revealed through single-molecule approaches

Helicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.

FORCE-FLUORESCENCE SPECTROSCOPY AT THE SINGLE-MOLECULE LEVEL

During the past decade, various powerful single-molecule techniques have evolved and helped to address important questions in life sciences. Yet these techniques would be even more powerful if they would be combined, that is, single-molecule manipulation with an orthogonal single-molecule observation. Here, we present a recently developed approach to combine single-molecule optical tweezers with single-molecule fluorescence spectroscopy. Optical tweezers are used to manipulate and observe mechanical properties on the nanometer scale and piconewton force range. However, once the force range is in the low piconewton range or less, the spatial resolution of optical tweezers decreases significantly. In combination with fluorescence spectroscopy, like Förster resonance energy transfer (FRET), we are able to observe nanometer fluctuations and internal conformational changes in a low-force regime. The possibility to place fluorescent labels at nearly any desired position and a sophisticated design of the experiment increases the amount of information that can be extracted in contrast to pure mechanical or fluorescence experiments.

Surprising Simplicity in the Single-Molecule Folding Mechanics of Proteins

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).

Complex Unfolding Kinetics of Single-Domain Proteins in the Presence of Force

Single-molecule force spectroscopy is providing unique, and sometimes unexpected, insights into the free-energy landscapes of proteins. Despite the complexity of the free-energy landscapes revealed by mechanical probes, forced unfolding experiments are often analyzed using one-dimensional models that predict a logarithmic dependence of the unfolding force on the pulling velocity. We previously found that the unfolding force of the protein filamin at low pulling speed did not decrease logarithmically with the pulling speed. Here we present results from a large number of unfolding simulations of a coarse-grain model of the protein filamin under a broad range of constant forces. These show that a two-path model is physically plausible and produces a deviation from the behavior predicted by one-dimensional models analogous to that observed experimentally. We also show that the analysis of the distributions of unfolding forces (p[F]) contains crucial and exploitable information, and that a proper description of the unfolding of single-domain proteins needs to account for the intrinsic multidimensionality of the underlying free-energy landscape, especially when the applied perturbation is small.

Folding of Proteins under Mechanical Force

Many proteins in the body are subject to mechanical forces in their natural context. Examples are muscle proteins or proteins of the cytoskeleton. Often a protein faces the challenge of refolding against a mechanical load. Although the unfolding of proteins under load has been extensively investigated, knowledge about refolding mechanics is still rare. This chapter develops a model that describes the effect of an external force on protein folding. The model can provide important help for the design of a single-molecule mechanical experiment. The chapter discusses how spacer length and elasticity, as well as probe spring constant, affect the observed results. In this context it also briefly discusses the difference between atomic force microscope and optical tweezers experiments.