DNA handles with biotin and digoxin are coupled to the N- and C-terminal cysteine residues of Cys-GB1-AFV3-Cys using the methods described in chapter 2 (2.2.2). GB1 is mechanically stable(149, 150) and would not be unfolded before the unfolding of AFV3-109, which we have proved in our AFM experiments in chapter 3 as we are pulling from N- and C-terminal of AFV3-109. Only the unfolding and refolding of AFV3-109 could be observed if the force is limited at low level (below 20 pN) and thus the mechanical unfolding and refolding of GB1 does not affect the analysis of our results. Figure 5.1 Representative force-extension curve of stretching and relaxation of AFV3-109 using optical tweezers.
The pulling speed is 20 nm/s. Light blue and light red traces are the raw data for stretching and relaxation at a sampling rate of 1 kHz, respectively. Traces filtered to 100 Hz are shown in dark blue and red. The pulling directions are indicated by arrows. The unfolding and refolding events are circled in blue and red, …show more content…
Indeed, our results from optical tweezers has demonstrated that AFV3-109 can successfully fold into slipknotted structure even against external force. As we described in Chapter 3, AFV3-109 has ~ 30 amino acid residues including β4 and β5 in threaded loop. The most difficult part of forming a slipknot is to insert the threaded loop into the knotting loop. The β1 and β2 stabilizes β4 and β5, respectively, which facilitates the process of loop insertion and reduce the energy barrier. However, AFV3-109 is a small protein and the topological hindrance can increase dramatically as the size of threaded loop increases. Further study on much larger slipknotted protein can provide more insights on the mechanism of the formation of