Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2.
We report single-molecule folding studies of a small, single-domain protein, chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for protein folding studies and has been extensively studied, both experimentally (at the ensemble level) and theoretically. Conformationally assisted ligation methodology was used to synthesize the proteins and site-specifically label them with donor and acceptor dyes. Folded and denatured subpopulations were observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopulations were directly monitored as a function of guanidinium chloride concentration. It is shown that new information about different aspects of the protein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency distributions are translated into potentials, and denaturation curves are directly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a destabilized mutant (K17G). Current limitations and future possibilities and prospects for single-pair FRET protein folding investigations are discussed.
Deniz
A A
AA
Department of Physics, University of California, Berkeley, CA 94720, USA.
Laurence
T A
TA
Beligere
G S
GS
Dahan
M
M
Martin
A B
AB
Chemla
D S
DS
Dawson
P E
PE
Schultz
P G
PG
Weiss
S
S
D004735:Energy Transfer; D019791:Guanidine; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D010940:Plant Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
D010446:Peptide Fragments; D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; C093336:chymotrypsin inhibitor 2; D019791:Guanidine