SYSTEM DEVELOPMENT FOR TIME-RESOLVED FRET IN SERCA

Abstract

We have compared mammalian (HEK) and insect (Sf21) cell expression systems for detecting structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA) by time-resolved fluorescence resonance energy transfer (FRET). X-ray crystallography suggests that calcium binding opens the cytoplasmic headpiece of SERCA, producing large increases in distance between nucleotide-binding, phosphorylation, and actuator domains (1.0–2.5 nm). To test this hypothesis using FRET, SERCA was labeled with cyan fluorescent protein (CFP) at the N-terminus in the actuator domain and fluorescein isothiocyanate (FITC) at Lys-515 in the nucleotide-binding domain. We expressed fluorescently-labeled SERCA in both HEK and Sf21 cells, purified endoplasmic reticulum membrane fragments from the cells, and then measured time-resolved FRET between CFP donor and FITC acceptor in the presence and absence of calcium. FRET determined that (1) the nucleotide-binding and actuator domain are closed in the calcium-free state and (2) calcium-binding induces a small opening (<0.2 nm) between these two domains. Similar results were detected by FRET in both mammalian and insect cell systems. We conclude that the cytoplasmic headpiece of SERCA is predominantly closed in biological membranes.