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. 1993 Jun;64(6):1750–1759. doi: 10.1016/S0006-3495(93)81546-7

Effect of Ca2+ binding on the profile structure of the sarcoplasmic reticulum membrane using time-resolved x-ray diffraction.

L J DeLong 1, J K Blasie 1
PMCID: PMC1262509  PMID: 8369405

Abstract

A number of studies have indicated that Ca(2+)-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 A in the membrane profile, but they also require decreased electron density within the cylindrically averaged profile structure of the Ca(2+)-ATPase immediately adjacent (< 15 A) 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.

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Selected References

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