Loading...
Detecting Intramolecular Dynamics and Multiple FRET States by Fluorescence Correlation Spectroscopy
Price, E. Shane DeVore, Matthew S. Johnson, Carey K.
Price, E. Shane
DeVore, Matthew S.
Johnson, Carey K.
Citations
Altmetric:
Abstract
Fluorescence correlation spectroscopy (FCS) is a robust method for the detection of intramolecular dynamics in proteins but is also susceptible to interference from other dynamic processes such as triplet kinetics and photobleaching. We describe an approach for detection of intramolecular dynamics in proteins labeled with a FRET dye pair based on global fitting to the two autocorrelation functions (green-green and red-red) and the two cross-correlation functions (greenred and red-green). We applied the method to detect intramolecular dynamics in the Ca2+ signaling protein calmodulin. Dynamics were detected on the 100-μs time scale in Ca2+-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro15-Cys) demonstrate the reliability of the method for isolating intramolecular dynamics from other dynamic processes on the microsecond time scale and confirm the absence of intramolecular dynamics of polyproline. We further show the sensitivity of the initial amplitudes of the FCS auto and cross-correlation functions to the presence of multiple FRET states, static or dynamic. The FCS measurements also show that the diffusion of Ca2+-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion.
Description
Date
2010-03-06
Journal Title
Journal ISSN
Volume Title
Publisher
American Chemical Society
Collections
Research Projects
Organizational Units
Journal Issue
Keywords
Calmodulin, Fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), Polyproline, Protein dynamics
Citation
Price, E. S., DeVore, M. S., & Johnson, C. K. (2010). Detecting Intramolecular Dynamics and Multiple FRET States by Fluorescence Correlation Spectroscopy. The Journal of Physical Chemistry. B, 114(17), 5895–5902. http://doi.org/10.1021/jp912125z