Rearrangement of the N and C terminal segments during the activation of the plasma membrane Ca2+-ATPase as revealed by intramolecular FRET between fused autofluorescent proteins

CORRADI GERARDO RAUL; ADAMO HUGO PEDRO

Ahrus, Dinamarca

Conferencia; 12th International ATPase Conference Na,K-ATPase and Related Transport ATPases of P-type: Structures, Mechanisms, and Roles in Health and Disease; 2008

Universidad de Ahrus

Rearrangement of the N and C terminal segments during the activation of the plasma membrane Ca2+-ATPase as revealed by intramolecular FRET between fused autofluorescent proteinsGerardo R. Corradi and Hugo P. Adamo Department of Biological Chemistry, Instituto de Química y Fisicoquímica Biologicas (IQUIFIB), Universidad de Buenos Aires, Junín 956, 1113 Ciudad de Buenos Aires, Argentina Correspondence to H.P.A.: hpadamo@qb.ffyb.uba.ar We have explored the possibility of obtaining a functional PMCA labeled with two autofluorescent proteins to be used as a reporter of conformational changes associated with the activity of the enzyme. The chimeric construct BFP-PMCA-GFP obtained by fusing the blue fluorescent protein after Thr2 and the green fluorescent protein at the C- terminal end of the human PMCA 4xb was successfully expressed in yeast and purified by calmodulin affinity chromatography. The BFP-PMCA-GFP performed similar to the wild type enzyme with respect to Ca2+-transport, Ca2+-ATPase activity and sensitivity to calmodulin activation. The fluorescence emission spectra of BFP-PMCA-GFP when the BFP fluorophore was excited at 387 nm showed an increase in the fluorescence intensity in the region of the spectra corresponding to the GFP emission. This was indicative of the existence of energy transfer between the two fluorescent proteins. In mixed micelles of detergent C12E10-phosphatidylcholine and at concentrations of BFP-PMCA-GFP lower than 25 nM the energy transfer occurred between fluorophores from the same molecule, and decreased when the enzyme was activated either by Ca2+-calmodulin, partial proteolysis, or acidic lipids. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation. This work was supported in part by the University of Buenos Aires (UBA, Grant B029), by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Grant PIP 6168), and by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Grant BID 1728 OC-AR PICT 15-25965).