title: 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 Ca(2+)-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 Ca(2+) binding sites. A high amplitude of dynamics in apo-calmodulin (in the absence of Ca(2+)) was nearly abolished at a high Ca(2+) concentration. For the C-terminal domain, the dynamic amplitude changed little with Ca(2+) concentration. The Ca(2+) 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 Ca(2+)-binding loops.
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
authors.forename: E Shane
authors.initials: ES
authors.lastname: Price
authors.forename: Marek
authors.initials: M
authors.lastname: Aleksiejew
authors.forename: Carey K
authors.initials: CK
authors.lastname: Johnson
mesh_terms: D002147:Calmodulin / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D013997:Time Factors
chemical_list: D002147:Calmodulin; D010455:Peptides; C011083:polyproline