Format

Send to

Choose Destination
Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):13021-6.

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy.

Author information

1
Departments of Chemistry and Biophysics and Biochemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.

Abstract

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

PMID:
11087856
PMCID:
PMC27171
DOI:
10.1073/pnas.97.24.13021
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center