CONFORMATIONAL CHANGES OCURRING DURING THE ACTIVATION OF THE PMCA BY CALMODULIN. L’TRAMOLECULAR FRET IN A BFP-PMCA-GFP FUSIO’ CONSTRUCT

GERARDO R. CORRADI; HUGO P ADAMO.

Bariloche Argentina

Simposio; SAIB PROTEIN SYMPOSIUM; 2003

A BFP-PMCA-GFP fusion protein suitable for intramolecular FRET measurements has been constructed by adding the blue (BFP) and green (GFP) fluorescent proteins at the N and C terminus of the plasma membrane Ca2+ transporter (PMCA). The recombinant protein purified from S. cerevisiae was a functional PMCA. When the purified protein was suspended in a media containing 500 μM EGTA and excited at 387 nm (BFP absorption maxima) the BFP-PMCA-GFP exhibited a mayor emission peak at 409 nm corresponding to BFP and a minor one at 509 nm due to GFP emission by FRET (Ida/Ia 0.2). Ca2+ did not significantly affect FRET. The removal of the fluorescent proteins from the PMCA by trypsin cleavage lead to a progressive disappearance of the peak at 509 nm (Ida/Ia 0.16). A similar drop in FRET was observed after addition of Ca-CaM. Adding enough EGTA to reduce the Ca2+ to less than 0.01 μM recovered Ida/Ia to a value close to that observed in the absence of CaM. These results are indicative of a conformational change occurring upon calmodulin binding and activation of the PMCA and suggest that the C terminal autoinhibitory region is moving apart or reorienting with respect to the N terminus which is part of the catalytic core of the enzyme.