Observation table:

Table 8.1: Observation table for the protein unfolding using fluorescence spectroscopy

Analysis of unfolding curve

For a two-state unfolding process, plotting the fluorescence intensity gives a curve as shown in figure 8.2. A least square fitting is performed on the data to obtain a continuous curve.

For a two-state folding/unfolding mechanism, only folded and unfolded protein states are populated at significant concentrations at any of the urea concentration. Therefore,

8.1

where, f_F and f_U represent the fractions of the folded and unfolded proteins, respectively.

Thus the observed value of y (fluorescence intensity, in this case) at any point in the graph is given by:

8.2

where, y_F and y_U represent the values of y characteristic of the folded and unfolded protein states, respectively and can be calculated from the unfolding curve (figure 8.2).

Combining equations 8.1 and 8.2:

8.3