Conformation changes in 2-Cys peroxiredoxin during catalytic cycle do not involve decamer disruption.

GERARDO R. CORRADI; HUGO P ADAMO

Montevideo Uruguay

Conferencia; 6º Conferencia internacional de Biofísica; 2007

Sociedad Argentina de Biofísica

The activity of the Ca2+ pump from human plasma membrane (isoform hPMCA4xb) is naturally low due to autoinhibition by its C-terminal region and its activity increases upon calmodulin binding to the autoinhibitory domain. The blue and green fluorescent proteins have been fused at the N and C terminal ends , respectively, of the plasma membrane Ca2+ pump isoform 4xb. The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins the BFP-PMCA-GFP performed similar to the wild type enzyme with respect to Ca2+-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state the BFP-PMCA-GFP exhibited a significant intramolecular FRET consistent with the location of the fluorophores at an average distance of  45 Å.. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca2+-calmodulin, partial proteolysis, or acidic lipids. Activation of hPMCA4xb by mutation D170N in  BFP-PMCA(D170N)-GFP decreased FRET and made it insensitive to calmodulin. These results indicates that in the autoinhibited state both ends of the PMCA are in close proximity ,and they separate  or  reorient  when the  PMCA achieves  its final activated conformation.