Time Resolved FRET in the SR Ca-ATPase

Abstract

We have detected structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA) using time-resolved fluorescence spectroscopy. The Ca-ATPase from fast-twitch skeletal muscle (SERCA1a isoform) was labeled with cyan fluorescent protein (CFP) at the N-terminus in the actuator domain (A) and fluorescein isothiocyanate (FITC) at Lys-515 in the nucleotide-binding domain (N). Time-resolved FRET was detected between CFP (donor in A domain) and FITC (acceptor in N domain) for SERCA in ligand-stabilized states, including calcium-free (E2), calcium-bound (E1), and actively-cycling phosphoenzyme (EP). Lifetime fitting and molecular modeling were used to interpret fluorescence decays, thereby identifying a dynamic distribution of structural states within the cytoplasmic headpiece of SERCA (i.e., A-N interdomain distance). FRET results were compared to distance predictions from x-ray crystallography. We propose a structural mechanism for ligand activation of SERCA. Acknowledgments: Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota, with assistance from Fluorescence Innovations, Inc. (Gregory Gillispie, President). This work was funded by NIH grants to DDT (R01 GM27906, P30 AR0507220, T32 AR007612).