Structural changes of sarcoplasmic reticulum Ca(2+)-ATPase upon Ca2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence.

Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca2+ from the photolabile Ca2+ chelator 1-(2-nitro-4,5-dimethoxy)-N,N,N',N',- tetrakis[(oxycarbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H2O and 2H2O were detected in the spectral region from 1800 cm-1 to 1200 cm-1, reflecting photolysis of DM-nitrophen and Ca2+ binding to the Ca(2+)-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca2+ binding activity of the ATPase in this specific step. Ca2+ 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 Ca2+ 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 1H2O/2H2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262-6267).