Orientation and rotational dynamics of spin‐labeled phalloidin bound to actin in muscle fibers

Naber, Nariman; Em, Ostap; Dd, Thomas; Cooke, Roger

doi:10.1002/prot.340170403

Cited by 8 publications

(10 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To confirm the binding stoichiometry of the rhodamine phalloidin-actin complex, actin was titrated with rhodamine phalloidin (Figure 2). When the free rhodamine phalloidin concentration was calculated assuming a stoichiometry of 1 actin monomer: 1 rhodamine phalloidin molecule (Cano et al, 1992;Huang et al, 1992;Naber et al, 1993), the Kf s obtained were 9 and 10 nM. This is in close agreement with the binding affinity determined from equilibrium experiments (Figure 1) and from the ratio of kinetic rate constants.…”

Section: Resultssupporting

confidence: 84%

See 1 more Smart Citation

Transient kinetic analysis of rhodamine phalloidin binding to actin filaments

Cruz

Pollard²

1994

Biochemistry

View full text Add to dashboard Cite

We have characterized the binding of rhodamine phalloidin to actin filaments and actin filaments saturated with either myosin subfragment-1 or tropomyosin in 50 mM KCl, 1 mM MgCl2 buffer at pH 7.0. Direct transient kinetic measurements of rhodamine phalloidin binding to actin filaments indicate an association rate constant of 2.8 x 10(4) M-1 s-1 and a dissociation rate constant of 4.8 x 10(-4) s-1. The ratio of the rate constants yields a dissociation equilibrium constant of 17 nM. From equilibrium measurements, the apparent affinity of rhodamine phalloidin for actin filaments is 116 nM. The difference between the affinities determined by equilibrium and kinetic experiments is attributed to the depolymerization of filaments at low actin concentrations in the equilibrium samples. The binding stoichiometry is one rhodamine phalloidin molecule per actin subunit. When myosin subfragment-1 and tropomyosin are bound to actin filaments, the rate constants for rhodamine phalloidin binding are the same as for actin alone and in agreement with the binding affinities measured in equilibrium experiments. Presumably these proteins stabilize the filaments. Neither substitution of CaCl2 for MgCl2 nor the inclusion of 20 mM phosphate altered the rate or equilibrium constants.

show abstract

Section: Resultssupporting

confidence: 84%

“…phalloidin (Figure 2). When the free rhodamine phalloidin concentration is calculated assuming a stoichiometry of 1 rhodamine phalloidin molecule per actin monomer (Wieland & Faulstich, 1978;Cano et al, 1992;Huang et al, 1992;Naber et al, 1993), the estimated K¿ s are 10.2 and 9.1 nM for the 50 and 100 nM actin data sets, respectively.…”

Section: Resultsmentioning

confidence: 99%

Transient kinetic analysis of rhodamine phalloidin binding to actin filaments

Cruz

Pollard²

1994

Biochemistry

View full text Add to dashboard Cite

show abstract

“…However, this does not exclude the possibility that cooperativity occurs across rather than along the actin filament. To test this, we added phalloidin, which binds near the thin filament axis with an orientation that is invariant with thin filament conformation (22) and both decreases thin filament flexibility and alters strand-strand interactions (23)(24)(25). Any cooperativity that was dependent upon such interactions might be changed by the addition of phalloidin.…”

Section: Relationship Between Ca 2ϩ Binding To the Thin Filament Regumentioning

confidence: 99%

Cooperative Effect of Calcium Binding to Adjacent Troponin Molecules on the Thin Filament-Myosin Subfragment 1 MgATPase Rate

Butters

Tobacman

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

The myosin subfragment 1 (S1) MgATPase rate was measured using thin filaments with known extents of Ca 2؉ binding controlled by varying the ratio of native cardiac troponin versus an inhibitory troponin with a mutation in the sole regulatory Ca 2؉ binding site of troponin C. Fractional MgATPase activation was less than the fraction of troponins that bound Ca 2؉ , implying a cooperative effect of bound Ca 2؉ on cross-bridge cycling. Addition of phalloidin did not alter cooperative effects between bound Ca 2؉ molecules in the presence or absence of myosin S1. When the myosin S1 concentration was raised sufficiently to introduce cooperative myosin-myosin effects, lower Ca 2؉ concentrations were needed to activate the MgATPase rate. MgATPase activation remained less than Ca 2؉ binding, implying a true, not just an apparent, increase in Ca 2؉ affinity. MgATPase activation by Ca 2؉ was more cooperative than could be explained by cooperativeness of overall Ca 2؉ binding, the discrepancy between Ca 2؉ binding and MgATPase activation, or interactions between myosins. The results suggest the thin filament-myosin S1 MgATPase cycle requires calcium binding to adjacent troponin molecules and that this binding is cooperatively promoted by a single cycling cross-bridge. This mechanism is a potential explanation for Ca 2؉ -mediated regulation of crossbridge kinetics in muscle fibers.Just as isometric tension is cooperatively activated by Ca 2ϩ , so is the cardiac thin filament-myosin S1 1 MgATPase rate, even under conditions where there is no cooperativity in myosin S1 binding (1, 2). A possible explanation for this behavior is that ATPase activation is proportional to Ca 2ϩ binding to the many TnCs on each thin filament and that this calcium binding is cooperative (3). We tested the idea that Ca 2ϩ binding and MgATPase activation are proportional and found to the contrary that they are not. Instead, fractional MgATPase activation was considerably less than fractional Ca 2ϩ binding and more closely paralleled the number of pairs of adjacent troponins with Ca 2ϩ bound to both.To accomplish the above experiment, we employ a constitutively inhibitory form of cardiac troponin containing an inactivating mutation of the sole regulatory site of TnC, site II (4). This troponin, designated CBMII-Tn, results in a low thin filament-myosin S1 MgATPase rate that is not increased by the addition of Ca 2ϩ , analogous to previous results in which a similar TnC mutant was exchanged into myofibrils or muscle fibers (5-7). CBMII-Tn binds to actin-tropomyosin with an affinity identical to that of normal troponin in the absence of Ca 2ϩ . This binding, which is very tight for both normal troponin and for 8,9), permits the present report in which thin filaments are assembled with defined mixtures of normal troponin and CBMII-Tn. In the presence of saturating Ca 2ϩ concentrations, such thin filaments exhibit a fractional saturation of the TnC regulatory sites that is experimentally controllable by varying the relative concentrations of the two forms ...

show abstract

“…bound between them. This tenuous connection could provide for flexibility between the outer two subdomains and the core of the filament; however, the existence of such a motion remains unproved.Although some investigators have proposed that dramatic conformational changes occur within the actin filament in the generation of force, data obtained from spin probes attached to thin filaments in muscle have not detected such changes(6). Spectroscopic studies have found that conformational changes do occur in actin upon binding of myosin, but none appear to be of the myosin head, shown inFig.…”

mentioning

confidence: 99%

The actomyosin engine

Cooke

1995

FASEB j.

Self Cite

View full text Add to dashboard Cite

Two recent advances have extended our knowledge of the actomyosin interaction considerably. The first of these is the determination of the crystal structures of the actin monomer and of the myosin head. The structures of these proteins were fit into the quaternary protein complexes of the actin filament, and of the actin filament decorated by the myosin head, using data from fiber diffraction or electron microscopy as constraints. The atomic models of these protein complexes have led to new hypotheses concerning the large conformational changes that must occur in these proteins in order to generate force. The second advance has been the measurements of the forces and displacements of single motor proteins, in particular, of a single myosin molecule interacting with a single actin filament. These data lead to more unambiguous interpretations than did previous mechanical data, which were obtained using ensembles of motors that worked asynchronously on their filaments. This approach brings to the motor field the equivalent of what patch clamp techniques brought to the study of membrane channels: the ability to look at the function of a single molecule.

show abstract

Orientation and rotational dynamics of spin‐labeled phalloidin bound to actin in muscle fibers

Cited by 8 publications

References 43 publications

Transient kinetic analysis of rhodamine phalloidin binding to actin filaments

Transient kinetic analysis of rhodamine phalloidin binding to actin filaments

Cooperative Effect of Calcium Binding to Adjacent Troponin Molecules on the Thin Filament-Myosin Subfragment 1 MgATPase Rate

The actomyosin engine

Contact Info

Product

Resources

About