EFFECT OF CA2+ BINDING ON THE PROFILE STRUCTURE OF THE SARCOPLASMIC-RETICULUM MEMBRANE USING TIME-RESOLVED X-RAY-DIFFRACTION

A number of studies have indicated that Ca2+-ATPase, the integral membrane protein of the sarcoplasmic reticulum (SR) membrane, undergoes some structural change upon Ca2+ binding to its high affinity binding sites (i.e., upon conversion of the E1 to the CaxE1 form of the enzyme). We have used x-ray diffraction to study the changes in the electron density profile of the SR membrane upon high-affinity Ca2+ binding to the enzyme in the absence of enzyme phosphorylation. The photolabile Ca2+ chelator DM-nitrophen was used to rapidly release Ca2+ into the extravesicular spaces throughout an oriented SR membrane multilayer and thereby synchronously in the vicinity of the high affinity binding sites of each enzyme molecule in the multilayer. A critical control was developed to exclude possible artifacts arising from heating and non-Ca2+ photolysis products in the membrane multilayer specimens upon photolysis of the DM-nitrophen. Upon photolysis, changes in the membrane electron density profile arising from high-affinity Ca2+ binding to the enzyme are found to be localized to three different regions within the profile. These changes can be attributed to the added electron density of the Ca2+ bound at three discrete sites centered at 5, approximately 30, and approximately 67 angstrom in the membrane profile, but they also require decreased electron density within the cylindrically averaged profile structure of the Ca2+-ATPase immediately adjacent (<15 angstrom) to these sites. The locations of these three Ca2+ binding sites in the SR membrane profile span most of the membrane profile in the absence of enzyme phosphorylation, in agreement with the locations of lanthanide (Tb3+ and La3+) binding sites in the membrane profile determined independently by using resonance x-ray diffraction.