Assessment
of the Folding Efficiency of Protein Domains in vitro
Adapted from: The European Working Group on CFTR Expression Resources
Authors: P.H. Thibodeau
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
It is well known that the structure and folding of a protein is encoded
by its primary amino acid sequence. This sequence defines both the three
dimensional native structure of the protein, and therefore its function, as well
as the folding pathway which enables a denatured protein to assume its compact
structure. The efficiency of folding of a protein is impacted by the
thermodynamic stabilities of the denatured, intermediate and native protein
populations as well the kinetics of both on- and off-pathway structure
formation. The aim of this protocol is to evaluate the macroscopic kinetic
properties of protein folding in a kinetic partitioning experiment that assesses
the competing reactions of productive folding, which leads to soluble protein, versus
non-productive misfolding, which leads to insoluble protein aggregates.
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. 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)
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 487.5ml of refolding buffer
(either with or without test compounds at 10mM final concentration)
into 1.5 ml Eppendorf tubes and incubate at the desired temperatures to
equilibrate the refolding buffer. Once the buffer temperatures have
equilibrated, add the denatured protein (12.5ml), vortex briefly (1
sec), and incubate at the desired temperatures overnight. After the refolding
reactions have completed, spin the tubes in a refrigerated microfuge at maximum
speed for 15 minutes at 4oC to pellet the misfolded insoluble
protein. Being careful not to disturb the pellet, remove the soluble protein
(supernatant) from the tubes for quantitative analysis by either SDS-PAGE or
fluorescence. In previous work, NBD1 refolded with high yield at 4°C (>95%
folded and soluble) while at temperatures in excess of 10-12°C refolding was
significantly less efficient (<50% folded and soluble).
REFERENCES
1.
Qu BH, Strickland EH, Thomas PJ. (1997) “Localization and Suppression of a
Kinetic Defect in Cystic Fibrosis Transmembrane Conductance Regulator
Folding.” JBC 272: 5739-15744.
2.
Qu BH, Thomas PJ. (1996) “Alteration of the Cystic Fibrosis Transmembrane
Condutance Regulator Folding Pathway: Effects of the F508 Mutation on the
Thermodynamic Stability and Folding Yield of NBD1.” JBC 271:
7261-7264.