Interdomain fluorescence resonance energy transfer in SERCA probed by cyan-fluorescent protein fused to the actuator domain.

ABSTRACT: We have used a biosynthetically incorporated fluorescent probe to monitor domain movements involved in ion transport by the sarcoendoplasmic reticulum Ca-ATPase (SERCA) from rabbit fast-twitch skeletal muscle. X-ray crystal structures suggest that the nucleotide-binding (N) and actuator (A) domains of SERCA move apart by several nanometers upon Ca binding. To test this hypothesis, cDNA constructs were created to fuse cyan-fluorescent protein (CFP) to the N terminus of SERCA (A domain). This CFP-SERCA fluorescent fusion protein retained activity when expressed in Sf21 insect cells using the baculovirus system. Fluorescence resonance energy transfer (FRET) was used to monitor the A-N interdomain distance for CFP-SERCA selectively labeled with fluorescein isothiocyanate (FITC) at Lys 515 in t

SUBMITTER: Winters DL 

PROVIDER: S-EPMC6537896 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature