Fluctuations in polarized fluorescence: evidence that muscle cross bridges rotate repetitively during contraction.

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...

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confidence: 59%

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confidence: 99%

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

Burghardt¹,

Ajtai²

1985

“…Because S-1 is elongate, a natural positional parameter is the angle that its "long" axis makes with the filament axis (4). There are now investigations showing that during isometric activity (i) this angle fluctuates (5) and (ii) the stationary value of this angle is quite different from what it is in rigor (6). Also there are investigations showing that (iii) in response to a step change in length a positional parameter of the cross-bridge relaxes (7).…”

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confidence: 99%

On the mechanism of energy transduction in myosin subfragment 1.

Botts¹,

Takashi²,

Torgerson³

et al. 1984

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It is proposed that the myosin subfragment 1 moiety of the muscle contractile apparatus is a self-contained "engine" whose operational plan is based on the interactive nature of ATP (or degradation intermediate) binding and actin binding, made possible by an intersite communication system. It is suggested that the spatial information required for examining this engine can, at least provisionally, be derived from fluorescence resonance energy transfer measurements interpreted by the Forster equation and that the existence of an intersite communication system can be deduced from piecewise detection of interacting pairs of points. A Theory of How the Myosin Subfragment 1 (S-i) Engine WorksIt has been thought that myosin S-1 moieties in active muscle fibers, pivoting about their attachments to subfragment 2 (S-2) moieties, deliver mechanical impulses to adjacent actin filaments, thus producing the relative interfilament translation ("slide") that characterizes isotonic muscle contraction (see ref. 1 for a review). Since there is no evidence that the gross shape (as distinct from local conformation) of S-1 changes during interaction with nucleotides (2) or actin (3), it is plausible to think that in its "purposeful" motion, as in its thermal motion, the cross-bridge rotates about S-1/S-2 as a quasirigid body. Because S-1 is elongate, a natural positional parameter is the angle that its "long" axis makes with the filament axis (4). There are now investigations showing that during isometric activity (i) this angle fluctuates (5) (7). Furthermore, the motional frequencies observed in i and iii are what might be expected from independent biochemical (8) and mechanical (9) observations and are magnitudes slower than thermally activated frequencies. Thus points i-iii support the idea that during activity cross-bridges move. They also suggest that the motion is describable by angles that position S-1 relative to the fiber axis (10), but in what follows it is necessary only that positional parameters relating S-1 to actin change with time repetitively.It is known that each S-1 bears an ATPase (N) site separate from the site (A) at which it binds actin (11) and that binding at these sites is interactive (12, 13). So it is tempting to think that the observed motions of S-1 are not those of an appendage passively moved by another agency, but rather that S-1 is a self-contained "unitary engine" strategically placed in the muscle apparatus. Previously we have elaborated on this hypothesis (14). We have noted that to examine this hypothesis we must look at common knowledge about S-1 in an uncommon way. For example, instead of considering S-1 motion in a fiber-based coordinate system we should position ligands (actin, nucleotides) in an S-1-based system, and we should think of enzymatic intermediates (15) as sequentially bound N-site ligands. These simple reinterpretations suggest a plan for the S-1 "engine": Because of the intersite communication system, binding of a particular intermediate at the N site determines a parti...

“…Relevant to our work are how the closely related methods of polarized fluorescence correlation spectroscopy (FCS) (17,18) and polarized f luorescence photobleaching recovery (PFPR) (19,20) were used to detect transient cross-bridge orientation (21,22). The polarized FCS from probe-modified cross-bridges in active isometric fibers detected for the first time the rotational movement of active cross-bridges, established their rotational relaxation time in isometric conditions, and confirmed the stochastic nature of their repetitive rotational movements (21). PFPR from similarly labeled crossbridges in active isometric fibers detected submillisecond cross-bridge relaxation rates and large probe angular displacement (22).…”

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confidence: 99%

Probes bound to myosin Cys-707 rotate during length transients in contraction

Burghardt

Garamszegi

Ajtai

1997

It is widely conjectured that muscle shortens because portions of myosin molecules (the ''cross-bridges'') impel the actin filament to which they transiently attach and that the impulses result from rotation of the cross-bridges. Crystallography indicates that a cross-bridge is articulatedconsisting of a globular catalytic͞actin-binding domain and a long lever arm that may rotate. Conveniently, a rhodamine probe with detectable attitude can be attached between the globular domain and the lever arm, enabling the observer to tell whether the anchoring region rotates. Well-established signature effects observed in shortening are tension changes resulting from the sudden release or quick stretch of active muscle fibers. In this investigation we found that closely correlated with such tension changes are changes in the attitude of the rhodamine probes. This correlation strongly supports the conjecture about how shortening is achieved.The molecular mechanism of muscle contraction involves the cyclical interaction of the myosin cross-bridge and the actin filament while myosin hydrolyzes ATP. Elucidation of this mechanism is sought at two levels, one local to the crossbridge-explaining how myosin transduces the free energy in ATP into the potential to do work (1)-and the other is at larger scale-explaining how the force to move myosin and actin filaments is generated (2-4). Experimentally, we need to detect and correlate local and global structural changes accompanying energy transduction and force generation in actomyosin. The spectroscopic probe techniques can give the required comprehensive ''two view'' description of the muscle contraction mechanism.An extrinsic fluorescent probe linked specifically to a side chain on the myosin cross-bridge or subfragment 1 (S1) emits a signal that can be detected and interpreted in terms of local and͞or global movement of the S1 (5, 6). The dual sensitivity of a probe can accurately reflect the local and global nature of contraction when both capabilities are exploited (7-10). For instance, by using a fluorescent probe, we recently investigated changes in the local conformation of the probe binding cleft of S1, a deep cleft containing the highly reactive cysteine (Cys-707 or highly reactive thiol, SH1) and a nucleotide-sensitive tryptophan 510 (Trp-510) (11), in response to ATP hydrolysis (12). † Alternatively, signals from several spectroscopic probes on SH1 in cross-bridges from fibers in steady-state conditions reported the global orientation of the cross-bridge in various physiological states including isometric contraction (6, 14-16). These global-orientation-detecting signals, when appropriately manipulated, combine to constrain fully a model for the cross-bridge angular trajectory during contraction (7,8).Time-resolved fluorescence experiments have similarly reported local and͞or global properties of the cross-bridge. Relevant to our work are how the closely related methods of polarized fluorescence correlation spectroscopy (FCS) (17, 18) and polarized f luorescence...