5.2.1 Comparison of the results from AFM and optical tweezers experiments
5.2.1.1 Different unfolding behaviors in AFM and optical tweezers experiments
In chapter 3, we have studied AFV3-109 using single-molecule AFM. The results from optical tweezers are contradictory to the results from AFM. First, the unfolding force obtained by optical tweezers is much smaller than the force obtained by AFM. More importantly, in our previous study using AFM, the unfolding of AFV3-109 occurs in two different pathways, two-state and three-state with an intermediate state at ~ 12 nm. However, in the results from optical tweezers, we did not observe any intermediate state. In addition, the unfolding distance of AFV3-109 measured by optical tweezers …show more content…
The force-extension traces were divided into many small time windows (Δt) that is small enough so that the force can be considered constant within a time window. The protein folding/unfolding rate can be calculated as P(F)=N(F)/M(F), where the N(F) is the total number of all the folding or unfolding events at the force of F and M(F) is the total number of time windows at the force of F. The probability of protein folding/unfolding within Δt at the force of F can be calculated as k(F)= P(F)/Δt.
5.3.4 Chemical Denaturation
Chemical denaturation experiments were carried out on a Cary Eclipse fluorescence spectrophotometer. The concentration of protein in fluorescence measurement was 0.2 mg/mL. The tryptophan fluorescence of protein at 350 nm was monitored at different concentration of guanidine hydrochloride. The result of fluorescence is fitted using the following equation: f(D) = ((aN+bN×D)+(aD+bD×D) ×exp(m× (D-D1/2)/0.592))/(1+exp(m× (D-D1/2)/0.592)) where D is the concentration of guanidine hydrochloride, aN and aD are the y-intercepts of native and denatured sections, respectively, bN and bD are the slopes of native and denatured sections, respectively, m is the slope of transition section and D1/2 is the concentration of guanidine hydrochloride at which half of the protein is in unfolded