Date of Award
August 2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Physics
First Advisor
Ionel IP Popa
Abstract
Unfolding and refolding of multidomain proteins under force is crucial for their function in vivo, in processes like mechanotransduction and muscle contraction. Understanding the dynamics of these proteins can have important implications in designing mechano-active drugs against conditions such as muscle dystrophy and cancer. Single-molecule force spectroscopy techniques such as magnetic tweezers allow for detailed observation of individual protein behaviors, revealing insights that are often hidden in ensemble measurements. Using stable tethers with magnetic tweezers, I measured single proteins over several hours at physiological force ranges to decipher their physical response to force. Polyproteins involved in regulating tissue elasticity and mechanotransduction unfold their domains under mechanical loads. While it is widely believed that the sequential unfolding of polyproteins follows an exponential kinetics with identical and identically distributed (iid) Poisson behavior, studies under high loads reveal non-exponential characteristics, suggesting aging due to subdiffusion. Statistical analysis shows these events are not iid, and do not follow a Poisson process. With numerical simulations, it was shown that this non-exponential behavior decreases with lower loads, indicating a potential return to Poisson behavior. This hypothesis was the motivation for the first project. We investigated the unfolding kinetics of polyprotein-L8 under varying loads, from high (100, 150 pN) to moderately-low (45, 30, 20 pN). Our findings reveal that there is hierarchy among unfolding events even under low loads, resulting in non-exponential behavior. I then followed these findings by investigating how protein molecules with similar structure might exhibit different responses. To this end, I designed and implemented several cross-linking chemistries between DNA and proteins. These constructs take advantage of a known transition of DNA at 65 pN, allowing for more accurate force calibration. The second project focused on talin, a force-sensing multidomain protein, which is a major player in cellular mechanotransduction. Using single-molecule magnetic tweezers, we investigated the mechanical response of the R8 rod-domain of talin. We observed that under various force cycles, the R8 domain of talin can display a memory-dependent behavior: at the same low force (<10 pN), the same protein molecule shows vastly different unfolding kinetics. This history dependent behavior indicates the evolution of a unique force-induced native state that can shift the binding affinity towards ligands. Indeed, we measured that talin R8 domain binds one of its ligands, deleted-in-liver-cancer-1 (DLC1), with much higher affinity than previously reported. This strong interaction can explain the antitumor response of DLC1, by regulating inside-out activation of integrins. Our results paint a more complex picture on the functioning of talin, which regularly unfolds and refolds under force, to produce a history-dependent response and fine-tune cellular focal-adhesions.
Recommended Citation
Sharma, Sabita, "Study of the dynamics of proteins at single-molecule level" (2024). Theses and Dissertations. 3623.
https://dc.uwm.edu/etd/3623