FRET-FCS Detection of Intralobe Dynamics in Calmodulin

Abstract

Fluorescence correlation spectroscopy (FCS) can be coupled with Förster resonance energy transfer (FRET) to detect intramolecular dynamics of proteins on the microsecond time scale. Here we describe application of FRET-FCS to detect fluctuations within the N-terminal and C-terminal domains of the Ca2+-signaling protein calmodulin. Intramolecular fluctuations were resolved by global fitting of the two fluorescence autocorrelation functions (green-green and red-red) together with the two cross-correlation functions (green-red and red-green). To match the Förster radius for FRET to the dimensions of the N-terminal and C-terminal domains, a near-infrared acceptor fluorophore (Atto 740) was coupled with a green-emitting donor (Alexa Fluor 488). Fluctuations were detected in both domains on the time scale of 30 to 40 μs. In the N-terminal domain, the amplitude of the fluctuations was dependent on occupancy of Ca2+ binding sites. A high amplitude of dynamics in apo-calmodulin (in the absence of Ca2+) was nearly abolished at a high Ca2+ concentration. For the C-terminal domain the dynamic amplitude changed little with Ca2+ concentration. The Ca2+ dependence of dynamics for the N-terminal domain suggests that the fluctuations detected by FCS in the N-terminal domain are coupled to the opening and closing of the EF-hand Ca2+-binding loops.

Direct Observation of T4 Lysozyme Hinge-Bending Motion by Fluorescence Correlation Spectroscopy

“…Although T4L hinge-bending motion has long been postulated (15), FCS enables a determination of its timescale in the range of~10-20 ms. Relaxation times in the range of microseconds to hundreds of microseconds have been reported for fluctuations arising from domain motions as well as transitions to partially unfolded states (30)(31)(32)(33)(34)(35).…”

“…Since its introduction in the early seventies (25), FCS has been used to examine a variety of systems including the photophysical behavior of fluorescent proteins (26,27) as well as macromolecular diffusion and interactions (28,29). In addition, FCS has been demonstrated to be a powerful tool for the investigation of equilibrium protein conformational and folding dynamics (30)(31)(32)(33)(34)(35). Hence, this technique is inherently suited to study the type of domain motion observed in T4L with the ultimate goal of obtaining a fluctuation timescale.…”

The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

“…For example BiFC has been used in FCS to investigate the recruitment of β-arrestin to NPY Y receptors [15]. FCS techniques have also been combined with FRET to investigate the dynamics of conformational changes of syntaxin-1 [34] and calmodulin [35]. …”

Single Pair Förster Resonance Energy Transfer: A Versatile Tool To Investigate Protein Conformational Dynamics

“…FCS measurements can be used to determine the timescale of conformational dynamics that are faster than the diffusion time. [124] Applications of this approach include the characterization of conformational dynamics of calmodulin, [130] diffusing biopolymers, [131] and glutamate receptor transitions. [132] In FRET-FCS, the signal of the donor is cross-correlated with the www.advancedsciencenews.com www.bioessays-journal.com acceptor channel.…”