This chapter discusses the functional effects of protein and lipid dynamics in sarcoplasmic reticulum. The chapter uses conventional electron paramagnetic resonance (EPR) of nitroxide spin labels to measure lipid chain rotational motions, to resolve lipid-protein interactions, and o devise a quantitative assay for lipid fluidity. It develops and uses both saturation transfer EPR (STEPR) and time-resolved phosphorescence anisotropy (TPA) to detect the global rotational diffusion of the protein. The chapter shows the way they have been combined and correlated with Ca-ATPase kinetics to assess the role of dynamic protein-lipid and protein-protein interactions in the enzymatic cycle. The spectroscopic probe techniques developed to measure and correlate the rotational motions of membrane proteins and lipids are outlined in the chapter. The two principal approaches are EPR and TPA that provides the means to observe rotational dynamics spanning the time scale from picoseconds to milliseconds. One of the unique aspects of the research described in the chapter is the use of both EPR and optical techniques in tandem, taking advantage of their complementary sensitivities.
Bibliographical noteFunding Information:
This work was supported by a National Institutes of Health grant (GM27906) to D.D.T, and J.E.M. was supported by a Postdoctoral Fellowship and a Research Grant-in-Aid from the American Heart Association, Minnesota Affiliate.