Structural changes of sarcoplasmic reticulum Ca 2+-ATPase upon Ca 2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence

Abstract

Ca 2+ binding to sacoplasmic reticulum Ca 2+-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca 2+ from the photolabile Ca 2+ chelator 1-(2-nitro-4,5-dimethoxy)- N, N, N′, N′,-tetrakis[(oxy-carbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H 2O and 2H 2O were detected in the spectral region from 1800 cm −1 to 1200 cm −1, reflecting photolysis of DM-nitrophen and Ca 2+ binding to the Ca 2+-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca 2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca 2+ binding activity of the ATPase in this specific step. Ca 2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm −1 can be assigned to Ca 2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm −1 to 6 cm −1 upon 1H 2O/ 2H 2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca 2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262–6267).