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

Abstract

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.

Rotational diffusion of a phosphor-labelled antibody bound to the human erythrocyte glucose transporter in inside-out vesicles from human erythrocyte ghosts

“…A2* exp (-t p 2 ) + A3. Rotation of this protein was very fast when compared to other membrane proteins like sarcoplasmic reticulum Ca-ATPase (Restall et al, 1984), the anion transporter of human erythrocytes (Muhlebach & Cherry, 1985) or rhodopsin (Coke et al, 1986), and the first time constant TI was usually below 5 ps, inside the region where interference from scattered light from the laser flash can occur. Fig.…”

“…An alternating conformation model has been proposed for the mechanism of the transport (Barnett et al, 1975;Lienhard et al, 1983), but little is known about the structural nature of this conformational change. The technique of time-resolved phosphorescence polarization has been used extensively to investigate the rotational dynamics of membrane proteins, particularly for the detection of conformational changes within proteins (Restall et al, 1984;Coke et al, 1986). Since existing methods do not usually allow the selective labelling of the protein under study, an important restriction in the study of protein rotational diffusion is the need for a purified membrane preparation.…”

Location and Properties of the Taxol Binding Center in Microtubules: A Picosecond Laser Study with Fluorescent Taxoids

“…The Flutax-Flutax distance estimated in this way and reported here is consistent only with a Taxol binding center located very close to the inner microtubule wall. isotropic solutions of fluorophores brought about by successive independent processes can be characterized by a series of depolarizing factors (33,34):…”

“…In this case, the overall depolarization is well approximated by where d T (t) is the depolarization factor for the overall free rotation of the taxoid as a whole and d L(T) (t) that for the hindered motion of the label with respect to the taxoid (34). P 2L is the Legendre polynomial of rank 2 for the restricted angular motion of the fluorophore with respect to the taxoid:…”

Quantitative fluorescence nanospectroscopy of nucleotide excision repair : from single molecules to cells

“…For example, there are five possible RCts for an elliptic macromolecule [226,227]. If a fluorophore label also experiences local depolarizing motions independent on the rotational motions taken by a macromolecule, the FA data analysis is compounded further because even more RCts must be resolved and interpreted [228][229][230][231]. As a result, FA can be dependent on the position of the fluorophore label on the macromolecule due to heterogeneous environment and complex depolarizing motions in many biomolecular system [231].…”