Rotational diffusion of calcium-dependent adenosine 5'-triphosphatase in sarcoplasmic reticulum: a detailed study

• Published in: Biochemistry (Easton) 1984-12, Vol.23 (26), p.6765-6776
• Main Authors: Restall, Colin J, Dale, Robert E, Murray, Elaine K, Gilbert, Charles W, Chapman, Dennis
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Ca(2+) Mg(2+)-ATPase; Calcium-Transporting ATPases; Conformational dynamics in molecular biology; Erythrosine - analogs & derivatives; Fluorescence Polarization; Fundamental and applied biological sciences. Psychology; Isothiocyanates; Models, Chemical; Molecular biophysics; Protein Conformation; Rabbits; Sarcoplasmic Reticulum - enzymology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00321a075

The Ca2+-Mg2+ adenosine-5'-triphosphatase (ATPase) in sarcoplasmic reticulum has been covalently labeled with the phosphorescent triplet probe erythrosinyl 5-isothiocyanate. The rotational diffusion of the protein in the membrane at 25 degrees C was examined by measuring the time dependence of the phosphorescence emission anisotropy. Detailed analysis of both the total emission S(t) = Iv(t) + 2IH(t) and anisotropy R(t) = [Iv(t) - IH(t)]/[Iv(t) + 2IH(t)] curves shows the presence of multiple components. The latter is incompatible with a simple model of protein movement. The experimental data are consistent with a model in which the sum of four exponential components defines the phosphorescence decay. The anisotropy decay corresponds to a model in which the phosphor itself or a small phosphor-bearing segment reorients on a sub-microsecond time scale about an axis attached to a larger segment, which in turn reorients on a time scale of a few microseconds about an axis fixed in the frame of the ATPase. A fraction of the protein molecules rotate on a time scale of 100-200 microseconds about the normal to the bilayer, while the rest are rotationally stationary, at least on a sub-millisecond time scale.