Format

Send to:

Choose Destination
See comment in PubMed Commons below
Methods Enzymol. 2010;472:41-60. doi: 10.1016/S0076-6879(10)72016-4.

Nanovesicle trapping for studying weak protein interactions by single-molecule FRET.

Author information

  • 1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.

Abstract

Protein-protein interactions are fundamental biological processes. While strong protein interactions are amenable to many characterization techniques including crystallography, weak protein interactions are challenging to study because of their dynamic nature. Single-molecule fluorescence resonance energy transfer (smFRET) can monitor dynamic protein interactions in real time, but are generally limited to strong interacting pairs because of the low concentrations needed for single-molecule detection. Here, we describe a nanovesicle trapping approach to enable smFRET study of weak protein interactions at high effective concentrations. We describe the experimental procedures, summarize the application in studying the weak interactions between intracellular copper transporters, and detail the single-molecule kinetic analysis of bimolecular interactions involving three states. Both the experimental approach and the theoretical analysis are generally applicable to studying many other biological processes at the single-molecule level.

Copyright 2010 Elsevier Inc. All rights reserved.

PMID:
20580959
[PubMed - indexed for MEDLINE]
PMCID:
PMC2992826
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science Icon for PubMed Central
    Loading ...
    Write to the Help Desk