Fraction of myosin cross-bridges bound to actin in active muscle fibers: estimation by fluorescence anisotropy measurements.

Abstract

Time-resolved and steady-state fluorescence anisotropy measurements from fluorescence-labeled myosin cross-bridges in single glycerinated skeletal muscle fibers in rigor, relaxed, MgADP-induced, and contracting states have been made in order to estimate the fraction of actin-bound cross-bridges in active muscle. When the plane of polarization of the excitation light is perpendicular to the fiber axis and its propagation vector has a component parallel to this axis, actin-bound cross-bridge states, such as rigor and MgADPinduced, have time-zero and steady-state anisotropies that are substantially lower than has the relaxed state. This difference provides a means of determining the fraction of cross-bridges bound to actin in active isometric fibers, by comparing the fluorescence anisotropy from active fibers with the anisotropy from bound and unbound cross-bridges in static states. By assuming that the active cross-bridges are either bound (in the manner of rigor or MgADP-induced states) or relaxed, we estimate that >80% of the cross-bridges are actin-bound in active isometric fibers.In the investigation of whether muscle myosin cross-bridges or parts of cross-bridges rotate during impulsion, extrinsic probes attached to the cross-bridges have been used because of their sensitivity to rotation. In our laboratory, work with fluorescent probes has generally confirmed the occurrence of rotational transitions between actin-attached states (1, 2), as well as indicated a high percentage (=65%) of cross-bridge attachment to actin during isometric tension generation (2). However, the work of others, using spin labels, has contradicted both results; in regard to percent attachment, estimates from spin-label work have indicated -20% attachment (3). The discrepancy concerning percent attachment, h, seems to us a good one on which to focus, since it is a quantity that can be estimated by various methods. In previous reports, estimates of h were based on angle information from iodoacetylrhodamine labels that reacted with the fast-reacting thiol (SH-1) of subfragment 1 (S-1) of myosin. We now report measurements of h obtained by similar reaction of SH-1 with 5-[2-(iodoacetyl)aminoethyl]aminonaphthalene-1-sulfonic acid (1,5-IAEDANS).Recent work in our laboratory has shown that, with 1,5-IAEDANS specifically attached to SH-1, thermal motion of cross-bridges in muscle fibers in the nanosecond time domain can be detected by use of the time-resolved fluorescence-anisotropy decay (TRFAD) technique as applied to ordered biological assemblies (4,5). In a previous TRFAD study, evidence was presented for two distinct rotational motions of the cross-bridge about two different axes of rotation (5). The distinction of the two cross-bridge motions is achieved by orienting the fiber in the excitation beam so that in one case (the "vertical fiber"), the polarization anisotropy is sensitive to cross-bridge motions about axes perpendicular to the fiber axis; and in another case (the "horizontal fiber"), the polarization anisotropy is sen...

Two-dimensional time-resolved X-ray diffraction studies of live isometrically contracting frog sartorius muscle

“…Based on our estimate of the limit of detectability of the heads still diffracting with a 14.3 nm spacing at peak tension, we conclude that 70%, or more of the heads are forming part of the AM complex at the plateau of tension. This is in agreement with figures for myosin head attachment based on the changes in intensity of the equatorial [I, 0, 0] and [1, 1, 0] reflections (Haselgrove & Huxley, 1973;Matsubara et al, 1975;Squire, 1981) and with the deduction from fluorescence depolarization investigations (Burghardt & Ajtai, 1985). It is also compatible with an interpretation of ESR data (Cooke et aI., 1982) which suggests that during isometric contraction approximately 80% of the probes are randomly oriented.…”

Energy, structure, conformation, and heart failure

“…High sensitivity of the above-mentioned motions to physiological fluctuations of Ca 2 § concentration [108,131] and the fact that these low-frequency motions (segmentary, rotational, and vibrational [84,115]) with microsecond rates are detected in functionally active protein regions (in C-terminal and with a some-what lower rate in N-terminal regions of actin polypeptide chain [52,131 ]) additionally confirm their role in force generation. Finally, the involvement of these motions in disturbances of myocardial contractility is confirmed by inhibition of nano-, micro-, and millisecond motions in actin protomer in HF caused by TAM, ATC, and LTC.…”

“…[107,115,129,131]), as well as from micro-to nanoseconds (motion of individual amino acid residues, and further to pico-and femtoseconds [58]. These high-frequency motions have no forcegenerating effect, but they underlie milli-and subnanosecond force-generating conformation transformations: there are direct correlations between subnanosecon movements of thin filaments in glycerinated muscle fibers and rigor tension [52] and between nanosecond motions in myosin (probably around the ATPase center) and generated force [129].…”

Myosin cross-bridge orientation in rigor and in the presence of nucleotide studied by electron spin resonance

“…In this case there was disagreement between findings using the probes 1,5-IAEDANS and IATR such that the former probe did not, while the latter probe did, sense a cross-bridge orientation change upon the binding of MgADP. It was suggested (Burghardt and Ajtai, 1985) and later confirmed experimentally (Ajtai and Burghardt, 1987) that this conflict was due to the differing orientations of the probes on the cross-bridge such that the transition dipole of IATR was in a favorable, while the dipole of 1,5-IAEDANS was in an unfavorable, orientation for detecting cross-bridge rotation. This explanation was confirmed using the technique of wavelength dependent fluorescence polarization where the transition dipole orientation of the 1,5-IAEDANS was changed, by varying the wavelength of the excitation light, and the cross-bridge rotation caused by the binding of MgADP was detected.…”