In conclusion, I performed both constant-velocity and constant-force AFM force spectroscopy experiments to probe single-molecule fibrin unfolding through the ‘A-a’ knob-hole interaction. From these experiments, I acquired new information about the complex forced unfolding pathway of fibrin’s γmodule including the effect of different solution conditions, previously unseen intermediate states, complex unfolding kinetics, and residue contacts responsible for experimentally observed unfolding events.
Constant-velocity experiments, designed to explore the relationship between solution environment and the single-molecule mechanics of fibrin interactions, revealed that the single-molecule force rupture pattern depend on solution conditions. Specifically, (1) a correlation was established between conditions that suppress the forced dissociation characteristic of the ‘A-a’ knob-hole interaction and those that have been previously reported to inhibit fibrin polymerization (i.e., high temperatures and acidic pH), and (2) a relationship
was found between solution conditions that caused a decrease in the probability of a characteristic ‘A-a’ interaction contained event 4 and those associated with fine fibrin clots with thin fibers. Therefore, the extension of the γ module provided by forces applied to the ‘A-a’ knob-hole interaction are essential for the formation and integrity of fibrin fibers and the extension provided by single fibrin molecules through extension of theγmodule has an impact on the size of fibers formed. Protofibrils have an inherent twist to them and may wrap around the fibrin fiber in order to form thicker fibers.(116; 137) The additional extension of theγ module could allow for the protofibrils to stretch permitting additional protofibril layers in thicker fibrin clots. In this way, SMFS experiments provide insight into the molecular mechanisms for larger scale clot behavior.
Constant-force experiments allowed for the investigation of low force unfolding domains and the direct investigation applied force’s affect on unfolding kinetics. This research marks the first constant-force un- folding/unbinding study performed on fibrin and more broadly on protein unfolding produced through a force application to a physiologically relevant bond. Through the application of low forces over a prolonged time period, I observed fibrinγ unfolding through previously unseen intermediate states. Analysis of un- folding kinetics revealed the fibrin γ module unfolds through a complex energy landscape best described by glassy dynamics behavior, characterized by stretched-exponential unfolding. Monte Carlo simulations revealed that force-clamp experiments measured a fundamentally different unfolding pathway than previous constant-velocity experiments. Other molecules, such as ubiquitin,(87) have shown evidence of intermediate states and deviations from a simple two-state unfolding model; however, subdomains were not implicated in the observed behavior. This work marks the first systematic study into the role of subdomain unfolding as the source of deviations from the unfolding.
DMD simulations were used to visualizeγmodule domains unfolding as a result of constant force appli- cation to the ‘A-a’ knob-hole interaction. Simulated fibrin unfolding experiments showedγmodule unfolding occurred through a series of steps - (1) reorientation of the protein along the axis of force, (2) separation of the ‘a’ binding pocket from theγ module, (3) formation of three subdomains, and (4) final unfolding of the γ module and formation of beta strands near the ‘a’ pocket. A variety of residues were implicated in unfolding events in the gamma module observed in simulation; however onlyγ285-90:319-24,γ301-5:382-3, γ 303-8:313-8, and γ 315-8:349-50 are likely to be observed in force-clamp experiments due to the their resulting extensions and location in theγmodule.
The combination of force-clamp experiments, Monte Carlo simulations and DMD simulations elucidated fibrinγmodule unfolding to a degree not previously achieved. The specific residues responsible for extension events observed in force-clamp experiments were identified through the use of DMD simulations. The exten- sion events associated with γ 285-90:319-24 and γ 301-5:382-3 produced∼3 nm extensions which together
produce the characteristic 6nm extension observed in force-clamp experiments. Monte Carlo simulations im- plicated the existence of multiple domains as the primary source of non-Markovian behavior observed in NEA analysis of force-clamp data. DMD simulations also showed multiple subdomains within theγ module, the earlier domains unfolding more rapidly than later domains. These simulations showed domains unfolding in order of their kinetic parameters (i.e., shorter lifetimes of conformational states earlier and longer lifetimes later) rather than the result of force-sheltered domains, which would produce short-lived conformational states following longer-lived conformational states. This level of detail into the mechanisms responsible for complexγmodule unfolding would not have been possible without the combination of these there methods. Preliminary experiments were performed to probe the reversibility of fibrinγmodule unfolding. Because fibrin fiber extensibility is largely reversible,(73) the molecular mechanisms responsible for fiber extension must also be reversible. If unfolding of the γ module induced by force applied to the ‘A-a’ interaction contributes to the extensibility of fibrin fibers, then it may play a role in the reversibility. A variation of force-clamp called force quench allows for investigation of protein refolding under applied force.(138; 121; 139; 140; 141) Force-quench works by applying a constant force for a period of time to unfold the protein (just like force-clamp), then reducing the force for a period of time to allow the protein to refold, and finally the larger unfolding force is applied again to observe the presence or absence of unfolding events.
I performed preliminary force-quench experiments in which a 100 pN force was applied for 0.5 seconds to induce some unfolding in theγ module, followed by a 1 second 10 pN force application, and finally another 100 pN force applied until rupture of the ‘A-a’ knob-hole interaction. These forces and time scales were selected based on force-clamp experiments to be most likely to induce folding but still maintain the ‘A-a’ knob-hole interaction. Two sample force-quench curves are shown in Figure 2.38. In order for force-quench curves to provide useful information into unfolding, the curve must possess the following characteristic: (1) the first high force application must induce an unfolding event, and (2) the knob-hole interaction must remain intact through the final high force application. Because fibrin unfolding is inherently a stochastic process, these criteria dramatically reduce the number of force curves in a population that provide insight into unfolding. For this reason only 20 force-quench curves were collected and examined. Preliminary results indicated that 15% of the force curves exhibit refolding behavior, characterized by an unfolding step in both the first and second high-force application, for the initially investigated parameters. The majority of force-quench curves did not exhibit refolding as evidenced by a second unfolding step; however, some of these force curves displayed complex separation traces during the force-quench interval. More experiments should be performed in which the quenching force is adjusted in order to observe refolding behavior.
This collection of experiments informed our understanding of the mechanical properties of fibrin as they relate to blood clot formation and protein unfolding in general. In the larger scope of designing
Figure 2.38: Representative force-quench results exhibiting both refolding and nonrefolding behavior of the γmodule. (Top) separation-time traces acquired for force-quench experiment exhibiting refolding (blue) and no refolding (red). The percentage of refolded and not refolded traces are reported based on a population of 20 force-quench curves. (Bottom) Force-time plots of the 100 pN, 10 pN and 100 pN force pulses of 0.5 s, 1 s, and 0.5 s for each of the force-quench experiments. Note, the red and blue force curves have been offset slightly in force to allow better visualization of force data.
an instrument for mechanical investigation of cells, these experiments provide an ideal system on which to develop force application experimental methods and analysis pipelines which can be applied to single- molecule measurements on cells. In the next chapter I will develop experimental methods and analysis pipelines necessary for measuring the mechanical properties of cells.
CHAPTER 3: Understanding the Role of Nuclear Mechanics Using the Atomic Force Microscope for Single-Cell Force Spectroscopy Studies
Living cells possess the ability to sense, withstand and respond to external mechanical forces. These properties are essential to the physical integrity and biological function of the cell. The molecular mechanisms responsible for a cell’s response to external force are especially of interest because of their downstream effects on gene expression, differentiation and motility. The nucleus itself has been implicated as a mechanosensor, with force-induced changes in nuclear structure directly affecting transcription; however, the process of mechanotransduction cannot be studied in isolation from cell mechanics.
The Atomic Force Microscope (AFM) has become ubiquitous in many cell mechanics studies, primarily due to its precision in force measurement (piconewtons), high spatial accuracy, and large dynamic range (hundreds of nanonewtons, depending on cantilever choice). In this chapter, I use the AFM to measure the mechanical properties of (1) two ovarian cancer cell lines with different known invasivity, (2) pancreatic cancer cells transfected with genes implicated in the epithelial-mesenchymal transition and (3) cytoplasts created by the removal of the nucleus from fibroblasts. Each of these studies provides insight into the role of nuclear mechanics, especially as it relates to mechanotransduction.
This chapter is comprised of the following key sections: