Fluorescence Depolarization of Actin Filaments in Reconstructed Myofibers: The Effect of S1 or pPDM-S1 on Movements of Distinct Areas of Actin

Abstract

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.

Orientation and Mobility of Actin in Different Intermediate States of the ATP Hydrolysis Cycle

“…[36]) and results of the present study (figure) indicate that weak and strong binding of myosin to actin is accompanied by different changes in orientation and mobility of the fluorescent probe. Change in number of myosin molecules influ enced only differences between the parameters studied under weak and strong binding.…”

“…FITC phalloidin is covalently bound to actin and so changes in the fluorescent probe orientation can be con sidered as changes in orientation of the whole protein molecule or its larger part [24,36,38]. The data obtained may be more easily interpreted by changes in azimuthal orientation of actin in thin filaments (e.g., by changes of actin monomer slope).…”

Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization

“…This model provides insight into how other actin-binding proteins, such as myosin (4,34), may take advantage of intrinsic multiple conformational states within F-actin.…”

“…Whereas actin was first isolated and studied as part of the contractile apparatus of vertebrate striated muscle (2), we now understand that actin is ubiquitous and plays an important role in many nonmuscle tissues. Within muscle, the traditional notion that actin is a passive cable on which myosin ''walks'' has been challenged by many observations (3)(4)(5)(6). Structural studies of actin filaments have revealed a surprising degree of internal plasticity, such as a variability in the twist (7,8) and tilt (9) of the component protomers, and these internal dynamics could play an important role in the interactions between actin and many other proteins.…”

Aberrant movement of β-tropomyosin associated with congenital myopathy causes defective response of myosin heads and actin during the ATPase cycle

“…Polarized fluorimetry has shown that in muscle fibers the switched on and switched off monomers differ in filament flexibility and orientation relative to the filament axis [13][14][15][17][18][19]. The amount of the switched on actin monomers in the filaments increases under the influence of TM, troponin-tropomyosin complex in the presence of Ca 2+ [13][14][15], S1, and NEM-S1 [20]. In contrast, troponin-tropomyosin complex in the absence of Ca 2+ [13][14][15], pPDM-S1 [20], and troponin I [21] decrease the number of the switched on subunits, i.e.…”