Conformational changes involved in the activation of the plasma membrane Ca2+ pump

CORRADI G. R; ADAMO H.P.

Congreso; 6ª Conferencia internacional de Biofísica; 2007

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.