of the Thermodynamic Stability
Authors: J.M. Richardson
and P.J. Thomas
Department of Physiology
The University of Texas
Southwestern Medical Center at Dallas
6001 Forest Park
Dallas, TX 75390-9040
The aim of this protocol is to measure the rate at which the off pathway
species accumulates from protein starting in the refolded state.
Protein in 8M GuHCL is rapidly diluted to conditions where refolding can
occur. The protein is then
allowed to refold at 4oC overnight.
Refolded protein is then increased in temperature to induce misfolding
and sample is monitored at 600nm for changes in light scattering.
As the off pathway species accumulates the aggregates scatter light
causing the signal to change as a function of time.
From this data a rate constant for the formation of off pathway species
can be extracted.
1. Purified protein
2. Refolding buffer (100 mM Tris, 375 mM Arginine, 2 mM EDTA, 1
mM DTT pH 7.9)
3. 8M Guanidine Hydrochloride (GuHCL)
4. Spectrophotometer with temperature control
5. Test Compounds
Purified protein is resuspended and incubated in 8M GuHCl for at least
two hours at room temperature to facilitate the complete denaturation of the
protein sample. Following denaturation, the protein concentration is determined
by UV absorbance. Adjust the protein concentrations of all samples to 40mM
by further dilution with 8M guanidinium such that both the guanidinium and
protein will be diluted by the same factor to achieve the final reaction
protein, and 200mM GuHCL) in all experiments. Both the protein and guanidinium
concentrations should be identical for all of the samples prior to and during
refolding. It is critical that all protein samples are at precisely the same
final concentration for accurate comparison in these experiments as protein
aggregation, the indicator for misfolded protein, is a concentration dependent
event. Aliquot 2437.5 ml
of refolding buffer into falcon tube with cap (without compounds) and
incubate at 4oC overnight. Sample
is then centrifuged to remove any misfolded protein for 15 min at 4oC.
Once any misfolded protein has been removed add test compounds to 10mM final concentration mix
quickly and incubate at 4oC for 70 minutes, then put the sample in a
spectroscopic cell with cap. Spectrophotometer
sample holder should be pre-equilibrated to desired temperature and ready to
start experiment. The sample is
induced to misfold at this concentration (1mM)
by increasing the temperature to either 32 or 37oC.
The increase in temperature causes the off-pathway species to become
populated, due to the equilibrium between the native and unfolded states. Stop
experiment when the refolding reactions have completed about 12,000 seconds or
when the UV signal reaches and maintains a plateau.