Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 24, Pages 8936-8941Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0401690101
Keywords
single-molecule fluorescence spectroscopy; forster resonance energy transfer; biomolecular interactions; catabolite activator protein; protein-DNA interactions
Categories
Funding
- NIGMS NIH HHS [R01 GM065382, GM65382:01] Funding Source: Medline
Ask authors/readers for more resources
We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of bionnolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Forster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available