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Mechanical unfolding and folding studies by optical tweezers Guo, Yabin


As a single molecule technique, optical tweezers technique proves to be a powerful tool to investigate the physical and chemical properties of DNA/RNA and protein molecules. In this thesis, optical tweezers are applied to two studies. In the first study, we directly investigated the unfolding and folding pathways and kinetics of the wild-type Top7 with optical tweezers. The existence of a folding intermediate state is confirmed. The unfolding process also occasionally shows non-cooperative behavior which has not been observed before. To identify if the mechanical stability of an isolated fragment of Top7 is responsible for the non-cooperative unfolding and folding behavior of Top7, we purified the C-fragment of Top7 and found that it reaches equilibrium at low applied forces, which indicates that Top7’s C-fragment could unfold and fold independently, but the unfolding and folding behavior of Top7 depends on the mutual assistance of both N-terminal and C-terminal residues. Illuminated by computational simulation methods, six residues were mutated aiming at improving the folding cooperativity of Top7. The results show that the folding cooperativity is improved significantly, while the unfolding intermediate appears more frequently. The possible influence of pathways on the frequency of occurrence of unfolding/folding intermediate state is discussed. In the second study, the two-step unfolding behavior of rubredoxin is revealed by optical tweezers. The reversible unfolding/folding behavior under force pressure and chemical pressure are further studied. Optical tweezers technique is proved to be well suited for mechanical unfolding/folding studies of metalloproteins.

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