Detecting intramolecular dynamics and multiple Förster resonance energy transfer states by fluorescence correlation spectroscopy.
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 the 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 (green-red and red-green). We applied the method to detect intramolecular dynamics in the Ca(2+) signaling protein calmodulin. Dynamics were detected on the 100 mus time scale in Ca(2+)-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro(15)-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 Ca(2+)-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion.
Price
E Shane
ES
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
DeVore
Matthew S
MS
Johnson
Carey K
CK
D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry*; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer; D010455:Peptides / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D010455:Peptides; C011083:polyproline; D002118:Calcium
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