Chevron Plot - Experimental Methodology

Experimental Methodology

To generate the folding limb of the chevron, the protein in a highly concentrated denaturant solution is diluted rapidly (in less than a millisecond) in an appropriate buffer to a particular denaturant concentration by means of a stopped flow apparatus. The relaxation to the new equilibrium is monitored by spectroscopic probes such as fluorescence or less frequently by circular dichroism (CD). The volume of the dilution is adjusted to obtain the relaxation rate at a specific denaturant concentration. The final protein concentration in the mixture is usually 1-20 µM, depending on the constraints imposed by the amplitude of relaxation and the signal-to-noise ratio. The unfolding limb is generated in a similar fashion by mixing denaturant-free protein with a concentrated denaturant solution in buffer. When the logarithm of these relaxation rates are plotted as a function of the final denaturant concentration, a chevron plot results.

The mixing of the solutions determines the dead-time of the instrument, which is about a millisecond. Therefore, a stopped-flow apparatus can be employed only for proteins with a relaxation time of a few milliseconds. In cases where the relaxation time is shorter than the dead-time of the instrument, the experimental temperature is lowered (thus increasing the viscosity of water/buffer) to increase the relaxation time to a few milliseconds. On the other hand, for fast-folding proteins (i.e., those with a relaxation rate of 1 to 100 microseconds), pressure-jump (dead time~few microseconds), temperature-jump (T-jump; dead time~few nanoseconds) or continuous flow mixing (dead time~few microseconds), can be carried out at different denaturant concentrations to obtain a chevron plot.