Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 May 9;97(10):5179-84.
doi: 10.1073/pnas.090104997.

Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2

A A Deniz  1 , T A Laurence, G S Beligere, M Dahan, A B Martin, D S Chemla, P E Dawson, P G Schultz, S Weiss
Affiliations

Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2

A A Deniz et al. Proc Natl Acad Sci U S A. .

Abstract

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
CI2 structure, showing points of dye attachment and destabilizing mutation.
Figure 2
Figure 2
(a) sp-FRET histograms of pwt CI2 at 3, 4, and 6 M denaturant. (b) sp-FRET histograms showing comparison of pwt and mutant CI2 (K17G) at 3.5 M denaturant. Solid lines show Gaussian fits to the peaks.
Figure 3
Figure 3
(a) Shift in the mean E of the denatured subpopulation for pwt CI2. The error bars are single SDs. The solid line is a guide to the eye. (b) Potentials V(R) (free energy functions) for pwt CI2 at 3, 4, and 6 M denaturant. The solid lines represent a smoothing of the data and are meant as a guide to the eye.
Figure 4
Figure 4
Ensemble and single molecule denaturation curves for pwt and mutant CI2. Symbols show data, lines show sigmoidal fits. Pwt CI2: ensemble tryptophan (○, solid line), ensemble FRET (□, dashed line), sp-FRET (×, dotted line); mutant CI2 sp-FRET (▿, dotted/dashed line).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dill K A, Shortle D. Annu Rev Biochem. 1991;60:795–825. - PubMed
    1. Smith L J, Fiebig K M, Schwalbe H, Dobson C M. Fold Design. 1996;1:R95–R106. - PubMed
    1. Brooks C L., 3rd Curr Opin Struct Biol. 1998;8:222–226. - PubMed
    1. Alm E, Baker D. Curr Opin Struct Biol. 1999;9:189–196. - PubMed
    1. Muñoz V, Eaton W A. Proc Natl Acad Sci USA. 1999;96:11311–11316. - PMC - PubMed

Publication types

LinkOut - more resources

  NODES
Association 1
twitter 2