Assessment
of the Misfolding Kinetics
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
email: philip.thomas@utsouthwestern.edu
INTRODUCTION
The aim of this protocol is to measure the rate at which the off pathway
species accumulates in a folding reaction starting in the unfolded state.
Protein in 8M GuHCL is rapidly diluted to conditions where refolding can
occur, sample is monitored at 600nm. 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.
MATERIALS
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
EXPERIMENTAL
PROTOCOL
Purified protein is 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 concentrations (1mM
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 3.0 ml spectroscopic cell with cap (either with or
without test compounds at 10mM
final concentration) and incubate at the desired temperature either 32 or 37oC
to equilibrate the refolding buffer. Once the buffer temperatures have
equilibrated, add the denatured protein (62.5ml),
mix briefly (1 sec), and start experiment. Stop experiment when the refolding
reactions near completed at about 12,000 seconds or when the signal reaches and
maintains a plateau.