FRET-FCS Detection of Intralobe Dynamics in Calmodulin

Fluorescence correlation spectroscopy (FCS) can be coupled with Forster resonance energy transfer (FRET) to detect intramolecular dynamics of proteins on the microsecond time scale. Here we describe application of FRET-PCS 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 Forster 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 mu s. In the N-terminal domain, the amplitude of the fluctuations was dependent on occupancy of Ca2+ binding sites. A high amplitude of dynamics in apocalmodulin (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.