Pulling single molecules of titin by AFM—recent advances and physiological implications
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Perturbation of a protein away from its native state by mechanical stress is a physiological process immanent to many cells. The mechanical stability and conformational diversity of proteins under force therefore are important parameters in nature. Molecular-level investigations of “mechanical proteins” have enjoyed major breakthroughs over the last decade, a development to which atomic force microscopy (AFM) force spectroscopy has been instrumental. The giant muscle protein titin continues to be a paradigm model in this field. In this paper, we review how single-molecule mechanical measurements of titin using AFM have served to elucidate key aspects of protein unfolding–refolding and mechanisms by which biomolecular elasticity is attained. We outline recent work combining protein engineering and AFM force spectroscopy to establish the mechanical behavior of titin domains using molecular “fingerprinting.” Furthermore, we summarize AFM force–extension data demonstrating different mechanical stabilities of distinct molecular-spring elements in titin, compare AFM force–extension to novel force-ramp/force-clamp studies, and elaborate on exciting new results showing that AFM force clamp captures the unfolding and refolding trajectory of single mechanical proteins. Along the way, we discuss the physiological implications of the findings, not least with respect to muscle mechanics. These studies help us understand how proteins respond to forces in cells and how mechanosensing and mechanosignaling events may proceed in vivo.
KeywordsAtomic force microscopy Titin Molecular mechanics Force–extension Force clamp Elasticity Protein folding
Mechanical forces are centrally involved in basic physiological processes, during both development and maintenance of normal function of differentiated cells and tissues. For example, estimates suggest that a newly synthesized polypeptide chain is exposed to an equilibrium force of ~10 pN as it exits the ribosomal tunnel . Physical stresses also play a role in various pathological states. A well-known example is the heart, which responds to mechanical (pressure or volume) overload with alterations in myocardial gene expression causing dysregulation of mechanical function and, eventually, cardiac failure. For an increasing number of researchers, it has become a major goal to understand the molecular and cellular basis of mechanotransduction in tissues. Along the same line, the growing field of mechanosignaling deals with elucidating the molecular mechanisms by which cells/tissues sense physical loads and transduce them into biochemical signals to alter gene expression and modify cellular structure and function.
The complex mechanotransduction network typically contains “mechanical proteins,” a term applicable to polypeptides designed to respond to force application under physiological conditions . To study the mechanical behavior of a mechanical protein, techniques are required that detect forces in the piconewton range and distances on the order of nanometers. Because of its versatility and relative ease of handling, the atomic force microscope has become the device of choice for many. A common method of mechanical manipulation by atomic force microscopy (AFM) is to pull a single molecule by a tip attached to a cantilever and probe the effect of stretch forces on protein stability. Mechanical forces are a natural protein denaturant, so the method reveals information that is likely to be of physiological relevance.
Apart from the study of how elastic proteins behave under stretch forces (covered in this review), AFM force spectroscopy has also been applied to measure adhesion forces , including those of individual proteins anchored in a membrane, like bacteriorhodopsin [47, 53, 100], analyze the binding forces between biotin and streptavidin  and the rupture forces of an antibody–antigen complex (termed “single-molecule recognition imaging microscopy”) [20, 43, 107, 125], or infer the strength of a covalent bond . Similarly, the so-called force–volume mode of the AFM can detect the presence of specific marker proteins on the surface of a cell by their binding strength to an antibody-coated AFM cantilever tip , a technique that distinguishes, for instance, red blood cells of group A from those of group B . Single molecules other than proteins have been deformed by pulling on them using the AFM; prominent examples include polysaccharides [84, 86, 111] and double-stranded deoxyribonucleic acid (DNA) [13, 18, 65, 113].
The “stretchy” titin segment: an adjustable molecular spring in muscle sarcomeres
Titin (also known as connectin ) is the largest known protein in nature. In humans, there is a single titin gene (on chromosome 2q31) coding for a total of 38,138 amino acid residues or a polypeptide with a maximum molecular mass of 4,200 kDa . Approximately 90% of titin’s mass is made up of globular domains of the immunoglobulin (Ig) or fibronectin-type-III (FN3)-like folds; the remainder is unique sequence insertions . An entire titin filament spans from the Z-disk to the center of the sarcomere, but the “stretchy bit” of titin is in the so-called I-band segment and varies greatly in length in different muscle types (Fig. 1b). Three main titin isoform classes with different I-band compositions are distinguished: N2B (~3.0 MDa) and N2BA (3.2–3.7 MDa) in the heart and N2A (~3.3–3.7 MDa) in skeletal muscles (Fig. 1b).
All isoforms express a “proximal” (I1–I15) and a “distal” (I84–I105) region composed of tandemly arranged Ig domains, as well as a “PEVK”-domain, named so for its high content in proline (P), glutamic acid (E), valine (V), and lysine (K) residues. Whereas an Ig domain comprises ~90–100 amino acids built in β-sandwich architecture , the PEVK domain contains coiled conformations that are elongated when the muscle is stretched [39, 40, 66, 72, 76, 92, 127]. Characteristic for the cardiac titin isoforms is a so-called N2-B region encompassing a long unique sequence (N2Bus) flanked by Ig domains. Cardiac N2BA and skeletal N2A isoforms additionally contain an “N2-A” domain and a central (“middle” or “variable-length”) Ig-domain region (I28–I79). Extensive alternative splicing occurring in this Ig domain region and in the PEVK domain (Fig. 1b) adjusts the contour length of the I-band titin  and is responsible for the great elastic diversity of the titin springs [94, 106]. The multiple functions of the titin proteins in muscle have been more extensively reviewed elsewhere [38, 78].
AFM stretching of engineered titin proteins: detecting single molecules by “fingerprinting”
Single-molecule AFM stretch experiments have been performed with isolated native titin [51, 110] or titin-like protein from invertebrate muscles . However, a great many studies have taken advantage of molecular engineering techniques to create recombinant constructs of various titin regions for examination by AFM [10, 15, 27, 35, 58, 63, 66, 67, 68, 85, 92, 97, 110, 112, 134, 135, 136]. Figure 1b (bottom of panel) lists all the parts of I-band titin that have, to our knowledge, so far been analyzed by AFM force spectroscopy using engineered fragments. Some of the constructs are multidomain proteins in which the modules are serially linked according to their natural sequence in the native molecule, e.g., (I24–I25-N2Bus-I26–I27) or (I91–I98) (Fig. 1c, upper two panels). Various other constructs are so-called polyproteins [16, 21] consisting of repeats of identical domains, e.g., (I4)8 or (I91-PEVKc)3 (Fig. 1c, lower two panels), which show unique mechanical features when stretched in AFM experiments .
In the actual AFM force–extension experiment, a drop of a protein suspension in physiological salt buffer is deposited onto a flat surface (a cleaned cover glass or mica, or a gold-coated glass surface), which can be moved with Angstrom accuracy by a piezoelectric actuator. When the tip of a flexible silicone nitride cantilever is brought in contact with the surface, sporadic binding can occur between the protein molecule and the tip atoms. If the other end of the molecule is immobilized to the surface, the protein can be stretched by increasing the distance between cantilever and surface (Fig. 1d). Forces in the range of a few tens of piconewtons up to several nanonewtons are measurable by detecting the deflection of the cantilever onto which the beam of a laser diode is focused. The reflected beam illuminates a two-segment photodiode, which records a differential signal that is fed into a feedback circuit (Fig. 1d). Because the spring constant of the cantilever can be precisely calculated using the equipartition theorem , the cantilever deflection is commensurable to the force applied to the protein.
Mechanical unfolding and refolding of titin Ig domains by force–extension
These data showed that Ig domain unfolding does not simply occur above a certain “threshold” force. Instead, it must be viewed as a stochastic process in which the unfolding probability is low (but above 0) in the absence of external force and increases with the applied force level to reach 100% at a force (Fmax) whose magnitude depends on the pulling speed. Results also suggested that most or all distal Ig domains in titin have high resistance to forced unfolding, whereas the proximal Ig domains are mechanically weaker , and their unfolding in response to elevated stretch forces is therefore more likely [75, 89]. However, the relatively high mechanical stability of these Ig modules rules out Ig domain unfolding as the principal mechanism by which titin springs respond to stretch forces in vivo.
What causes the resistance to unfolding in titin Ig-domains? An invaluable contribution to understanding the critical events leading to mechanical unfolding has come from steered molecular dynamics (SMD) simulations and other modeling approaches [4, 28, 31, 32, 54, 80, 81, 85, 103, 104, 137], which have used the available atomic structures of the titin Ig domains I1  and I91 . For I91, SMD simulations showed that mechanical force application first breaks two backbone hydrogen bonds between the beta-strands A and B—an event that is sometimes projected in AFM recordings as a small “hump” during the force rise (see Figs. 2b and 3a) [41, 48, 67, 85]. This unfolding intermediate state is followed by the breakage of additional hydrogen bonds between the beta-strands A′ and G to cause complete unfolding. In comparison to I91, the Ig domain I1 shows a simpler two-state transition during unfolding [32, 67, 68]: The backbone hydrogen bonds between beta-strands A–B and A′–G rupture simultaneously, and the unfolding of I1 occurs in an all-or-none fashion. A twist in the story is that the I1 domain can form a disulfide bridge between beta-strands C and E, which could stabilize this domain under oxidative stress conditions in muscle cells [68, 88]. In sum, comparisons of AFM data with SMD simulation studies have provided us with atomic-level insights into the events taking place during force-induced unfolding of a titin Ig domain. Titin has served as an important model for understanding key issues of protein unfolding.
The Ig domains unfold reversibly. Refolding occurs spontaneously in physiological salt buffer upon lowering the stretch force level; misfolding is a rare event  despite the absence of chaperones. Reversible unfolding of titin has been studied by AFM force–extension using a double-pulse protocol, where the protein is first completely unfolded but not allowed to detach from the cantilever, so that it can be relaxed again and—after a variable waiting period—stretched a second time [11, 15, 97, 110]. The rate of refolding is calculated based on the percentage of domains that have folded during the waiting time, which can be determined by counting the unfolding peaks in the second stretch pulse. For titin I91, refolding rates of ~1 s−1 at room temperature were found , whereas faster rates up to 15 s−1 at 25°C were recently reported for titin-like Ig/FN3 domains from invertebrate muscle . The observation that titin Ig domains can be unfolded in a reversible manner raises the possibility that this mechanism is of importance in vivo. This issue will be discussed in more detail below in the context of refolding studies using AFM force clamp. Reversible unfolding of Ig domains may in part explain the hysteresis appearing in stretch–release cycles of nonactivated sarcomeres [50, 70, 89, 129].
Force–extension studies demonstrate different mechanical stabilities of the molecular spring elements in titin
AFM force–extension experiments on engineered polyproteins have been extremely useful for understanding the molecular mechanisms of titin elasticity in vivo. To this end, the generation of heteropolyproteins (Fig. 1c) has been instrumental [66, 67]. Concatemers of the type (I91)3-N2Bus-(I91)3 [67, 75] or (I91-PEVKc)3 [66, 67, 76] essentially represent synthetic “mini-titins” exhibiting mechanical features (Fig. 3b,c), which one also expects in the full-length titin. In these heteropolyproteins, the I91 Ig domains again serve as the mechanical fingerprint to detect a single-molecule tether. A typical force–extension curve of (I91)3-N2Bus-(I91)3 shows a long low-force region before the first unfolding peak, corresponding to the extension of the N2B-unique sequence (Fig. 3b). In this example, the tether contained four I91 Ig modules (four unfolding peaks spaced at 28 nm) plus the whole intervening N2Bus, which according to WLC fits (blue curve in Fig. 3b) had a persistence length, A, of 0.7 nm and a contour length, L, of ~200 nm. The latter value matches the contour length expected for a fully extended N2Bus of 572 residues [67, 75]. A representative force trace for (I91-PEVKc)3 also shows an initial low-force region during which PEVK extension occurs, followed by two Ig-unfolding peaks spaced at 28 nm (Fig. 3c). Thus, the tether contained two I91 Ig modules and at least one full PEVK segment of 186 residues (this PEVK domain is from the cardiac N2B isoform). In this example, the persistence length for the PEVK region was 1.0 nm, and the contour length was ~80 nm. One conclusion drawn from these experiments is that the unique sequences in titin generate elastic forces mainly according to an entropic-spring mechanism, although evidence abounds suggesting that additional factors determine the elasticity of the PEVK domain [27, 58, 72, 76, 92, 132, 137]. In any case, these and related studies [27, 58, 62, 66, 67, 92, 135] clearly demonstrated that the PEVK domain and the N2Bus extend at forces at which the Ig domain unfolding probability is still very low.
If one compares the WLC parameters for the distinct molecular-spring elements in titin, it becomes immediately obvious that these elements have different mechanical stabilities. The WLC model (Eq. 1) predicts that the force needed to extend a polymer chain critically depends on the persistence length, A: The smaller the persistence length, the higher is the force. Therefore, upon stretching titin, the PEVK domain (A ~ 1 nm) will begin to unravel before the N2Bus (A ~ 0.7 nm) does . However, even before these unique sequences extend, there will be a straightening out of titin’s Ig domain regions because those regions have a much longer persistence length of greater than or equal to 10 nm [67, 73, 75, 130]. Taken together, the AFM data have allowed for a prediction of how the different titin segments extend in the sarcomere (Fig. 3d). Beginning from slack sarcomere length, low stretch forces will first straighten out titin’s proximal and distal tandem Ig regions. Once the extensibility of the (folded) Ig domain regions is largely exhausted, the unique sequences will begin to extend, first the PEVK domain and second (only in heart muscle) the N2Bus [67, 135]. This model essentially confirmed previous ideas about titin segment extension in vivo proposed on the basis of in situ measurements of titin extensibility using immunolabeling techniques [34, 71, 72, 74, 127].
Notably, owing to the coexpression of N2B and N2BA isoforms in the half-sarcomeres of cardiac muscle (Fig. 3d), the fractional extension (x/L) in situ is much higher for the short N2B than for the longer N2BA I-band segment. Within the physiological sarcomere-length range in the heart (~1.8–2.4 μm), the longer N2BA springs will only straighten out their Ig regions but not unravel the unique sequence insertions (Fig. 3d). Therefore, at a given sarcomere stretch state, the N2B isoform will be stiffer than the N2BA isoforms. Nature in fact uses alterations in the composition of stiff vs compliant titin isoforms to adjust the passive stiffness of the cardiac myocytes both during fetal/perinatal development [57, 60, 102, 133] and in chronic human heart disease [82, 91, 93]. The AFM studies have greatly helped us understand the molecular basis for these stiffness adjustments.
Novel approaches to studying unfolding–refolding by AFM: force ramp and force clamp
In the unfolding staircase during force clamp, each step marks the unfolding dwell time, t, which is defined as the time it takes for each module in the chain to unfold, measured from the time point the force is applied (Fig. 5e). Recording the dwell times for a large number of individual unfolding events allowed for a statistical analysis of the system’s kinetics [9, 33, 98]. Measurements showed that for a polyprotein of titin I91, the dwell-time distribution follows a single exponential (although significant deviations exist hinting at a more complex energy landscape during unfolding [8, 9, 98]), suggesting that the mechanical unfolding events in the polyprotein are independent of the unfolding history . These data implied that there is no mechanical coupling between unfolding Ig domains. Recent work comparing the unfolding of polyproteins and monomers of I91 under force clamp  has confirmed this view: The modules unfold independently of one another.
The usefulness of the force-clamp mode is further illustrated by the fact that one can straightforwardly deduce the unfolding probability, Pu, of the Ig domains (without requiring Monte Carlo simulations as in the case of force–extension data) by fitting a single exponential to the length-vs-time trajectories, as done in Fig. 5f; a few averaged trajectories are sufficient to obtain Pu. This analysis has been done for (I91)8 unfolding under different clamp forces from 80 to 200 pN . The rate constant for unfolding, α, obtained from exponential fits like the one shown in Fig. 5f, was found to be exponentially dependent on the applied force. Plotting the logarithm of α against the clamp force and fitting a straight line to the data using Eq. 2 (the Bell model; Fig. 5f, inset) yielded an unfolding rate at zero force, α0, of 7.2 × 10−4 s−1 , close to the value found in force–extension studies on I91. The results were similar for the polyprotein (I91)8 and monomers of I91 suggesting that nonspecific interdomain or domain–surface interactions potentially influencing the unfolding are likely to be absent. A general conclusion drawn from these studies is that unfolding at a given force is stochastic and may follow a simple two-state (Markovian) kinetic process, whereas the rate of unfolding increases exponentially with the force level.
Taken together, the unfolding measurements under force clamp have provided a faithful description of the mechanical stability of modular proteins and deepened our knowledge about how mechanical proteins like titin behave under external force. It is not unreasonable to expect that these experiments will eventually turn out to be extremely useful for understanding how proteins respond to mechanical forces in the cell and how mechanosensing or mechanical signaling are accomplished in vivo.
Refolding of titin Ig domains under force
The original conclusion that the folding may be cooperative between the modules in the polymer chain —unlike the stochastic, two-stage process observed for the unfolding—triggered discussions on how to interpret the force-clamp data. It was suggested that the apparent cooperativity could be due to aggregation between the neighboring domains within the polyprotein  or to a masking of refolding steps by large thermal fluctuations of the unfolded polyprotein . However, these interpretations did not hold up in light of the fact that monomers of ubiquitin or titin I91 show a collapse/refolding behavior, which is very similar to that of the multimodule proteins, demonstrating that the folding pathways are not affected by the presence of neighboring domains . Modeling studies on I91 also suggested that the refolding pathways are genuinely heterogeneous , a proposition confirmed by experiment . One can conclude that a mechanically unfolded protein is likely to first collapse from an extended state to a “molten globule,” a state in which it dwells for a variable lifetime, before it actually refolds. These findings and conclusions are remarkable because they imply that the energy landscape explored during refolding of a protein that has been mechanically denatured is different from that explored during refolding of a thermally or chemically denatured protein [69, 132], which until recently was the only way to study protein folding (usually done in bulk biochemistry experiments). AFM force clamp has thus offered unprecedented glimpses into physiologically relevant mechanisms driving protein folding.
Perturbing a protein away from its native state by mechanical force is a natural process, as many cells live in an environment that is under mechanical stress . Therefore, the mechanical stability and conformational diversity of a protein under force are important parameters in nature . These parameters are readily determinable by single-molecule force clamp. Just like the statistical analysis of single-molecule kinetics of biological reactions has revealed the mechanisms of important processes—e.g., the function of ion channels in cell membranes [19, 42], the evoked synaptic transmission in neurons , or the contraction of muscle cells —the results obtained in AFM force-clamp measurements are likely to have a great impact on how we will view the function of mechanical proteins in vivo.
Titin Ig domain refolding: implications for understanding titin mechanics in muscle
The force-clamp experiments directly proved that Ig domains can refold within seconds against significant stretching forces (Fig. 6) of less than or equal to 30 pN (titin-like proteins from insect muscle) [11, 61] or less than or equal to 25 pN (titin I91) . These findings confirmed and extended previous reports demonstrating that refolding can occur even when the protein is not fully relaxed [49, 110]. Some time ago, it was proposed that individual titin Ig domains unfold reversibly in vivo to provide the necessary extension during stretch of muscle sarcomeres [22, 122]. However, the issue of whether unfolding and refolding take place in muscle is still unresolved. Based on experimental and modeling studies on isolated myofibrils, it was concluded that massive Ig domain unfolding cannot happen in sarcomeres, whereas a few Ig domains per titin molecule could nevertheless unfold in response to rapid stretching . Ig domain unfolding was suggested to contribute to the phenomenon of stress relaxation in stretched nonactivated muscle fibers, i.e., force decay at constant length. Single molecules of titin did indeed show stepwise force relaxation when mechanically manipulated by optical tweezers . Recent evidence also suggested that FN3 domains in fibronectin, which readily unfold in AFM force spectroscopy measurements, unfold in native extracellular matrix fibrils, as well .
In this paper, we offer a simple explanation why under physiological conditions in relaxed human soleus muscle fibers Ig domain unfolding was not detectable . If titin Ig domains unfold in the sarcomere but also refold rapidly under relatively high forces, a model of the kind shown in Fig. 7 can be proposed: Upon stretch, a few Ig domains per titin strand may unravel, but even during extended waiting times in the stretched state, an antibody to the N2-A epitope would remain stationary relative to the Z-disk because the initially unfolded domains will refold (arrowheads in Fig. 7, bottom panel), while other modules unfold. Eventually, there will be a dynamic equilibrium between unfolding and refolding. This testable model could be used as a working hypothesis to further examine the possibility of Ig domain unfolding in vivo. If Ig unfolding–refolding did occur in sarcomeres, it could act as a shock absorber mechanism to help prevent irreversible damage to the muscle cells during application of elevated stretch forces.
What started a decade ago as a brilliant idea to mechanically manipulate titin filaments and engineered titin constructs using the AFM  has developed into a full-fledged field investigating important issues of force-induced protein unfolding and refolding in many different mechanical proteins or other mechanically active molecules. This field has long matured beyond the phenomenological and now addresses a variety of important biological questions, including, e.g., the relationship between protein structure and response to external force or the relevance of single-molecule data to physiological mechanisms. Titin remains a focus of attention, as well. Studies on the reversible unfolding of titin Ig domains continue to teach us important lessons about the response of mechanical proteins to stretching forces and the mechanisms by which biomolecular elasticity is accomplished.
An important issue in future work will be how conditions in AFM force spectroscopy experiments could be adapted to better mimic the physiological setting. This might include: realizing single-molecule force measurements in a protein’s natural environment [47, 53], optimizing ambient conditions (be aware, for example, of the viscosity and the reducing environment in cells ), engineering and investigating multimodular proteins in parallel  to obtain well-oriented homotypic aggregates, which are frequently found in vivo , or studying alterations in single-molecule mechanical properties induced by interaction with a ligand (e.g., a chaperone)  or phosphorylation by a protein kinase. The latter could turn out to be extremely useful for understanding mechanosensing and mechanosignaling events, which are of special importance not only in muscle cells, at the submolecular level. To this end, single-molecule AFM force spectroscopy may be combined with single-molecule fluorescence imaging techniques (e.g., total internal reflection fluorescence microscopy or fluorescence resonance energy transfer) to detect mechanical and biochemical signaling events at the same time. This technically challenging task has already revealed promising results [52, 116] but requires further refinement. A different line of research will be directed at employing AFM force spectroscopy in combination with protein engineering techniques to create protein-based advanced materials with novel mechanical properties . Finally, by combining experimental and theoretical (simulation) approaches , the exciting and expanding field of mechanotransduction will be on a faster track to reach its ultimate goal: a better understanding of the mechanisms by which cells sense and process mechanical information.
We thank Dr. Sergi Garcia-Manyes for providing the force trajectory of (I91)8 shown in Fig. 5 and Drs. Lorna Dougan, Hongbin Li, Andres Oberhauser, and Prof. Julio Fernandez for critical reading of the manuscript. We also acknowledge financial support by the DFG (SFB 629).
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