Excimer Formation of ATPase from Sarcoplasmic Reticulum Labeled with N‐(3‐Pyrene)maleinimide

Abstract

Sarcoplasmic reticulum ATPase froin fast skeletal muscle was labeled in native vesicles with N-( 3-pyrene) maleininiide. At labeling ratios larger than 1 mol pyrenemaleinimide/2.5 mol ATPase significant amounts of excimers are detected. Excimer concentration decreases at low, non-solubilizing amounts of detergents (0.2 mg . iiig protein-') and completely disappears after solubilization of the membranes. These results exclude that excimers are formed due to 'double-labeling' of one ATPasc molecule. It is concluded that the ATPase exists as an oligoiner within the membrane of native vesicles. The investigation of protein-protein interactions in membranous systems ideally requires a 'reporter group' with a short lifetime ( < 1 ps) (if compared to the rotation and diffusion of proteins) which is sensitive to small distances ( < 1 nm). This conditions appear largely fulfilled by proteinbound pyrene labels.Pyrene was shown to form excimers at high concentrations in organic solvents [25] and also in the I-butanethiol adduct of pyrenemaleinimide [26]. The distance between the two pyrene molecules is about 0.3 nni [27] and the pyrene excimers have a lifetime of about 50 ns [28]. The applicability of the pyrcne labcl requires that the proteins can be selectively labeled with a pyrene derivative.Sarcoplastnic reticulum vesicles labeled with N-(3-pyrene)-inaleinimide indeed show a significant amount of excimers at quite low label concentrations (1 mol labe1/2.5 mol ATPase). Since excimers were not due to double labeling of one ATPase molecule the results strongly suggest an oligomeric form of the ATPase within the membrane.