Biomedical subjects

Ewa Prochniewicz

Publications and source records attributed to Ewa Prochniewicz.

5 recordsLinked to original sources

PubMed2005

Structural dynamics of the actin-myosin interface by site-directed spectroscopy.

We have used site-directed spin and fluorescence labeling to test molecular models of the actin-myosin interface. Force is generated when the actin-myosin complex undergoes a transition from a disordered weak-binding state to an ordered strong-binding state. Actomyosin interface models, in which residues are classified as contributing to either weak or strong binding, have been derived by fitting individual crystallographic structures of actin and myosin into actomyosin cryo-EM maps. Our goal is to test these models using site-directed spectroscopic probes on actin and myosin. Starting with Cys-lite constructs of both yeast actin (ActC) and the Dictyostelium myosin II motor domain (S1dC), site-directed labeling (SDL) mutants were generated by mutating residues to Cys in the proposed weak and strong-binding interfaces. This report focuses on the effects of forming the strong-binding complex on four SDL mutants, two located in the proposed weak-binding interface (ActC5 and S1dC619) and two located in the proposed strong-binding interface (ActC345 and S1dC401). Neither the mutations nor labeling prevented strong actomyosin binding or actin-activation of myosin ATPase. Formation of the strong-binding complex resulted in decreased spin and fluorescence probe mobility at all sites, but both myosin-bound probes showed remarkably high mobility even after complex formation. Complex formation decreased solvent accessibility for both actin-bound probes, but increased it for the myosin-bound probes. These results are not consistent with a simple model in which there are discrete weak and strong interfaces, with only the strong interface forming under strong-binding conditions, nor are they consistent with a model in which surface residues become rigid and inaccessible upon complex formation. We conclude that all four of these residues are involved in the strong actin-myosin interface, but this interface is remarkably dynamic, especially on the surface of myosin.

Actins↗

PubMed2005

Cofilin increases the torsional flexibility and dynamics of actin filaments.

We have measured the effects of cofilin on the conformation and dynamics of actin filaments labeled at Cys374 with erythrosin-iodoacetemide (ErIA), using time-resolved phosphorescence anisotropy (TPA). Cofilin quenches the phosphorescence intensity of actin-bound ErIA, indicating that binding changes the local environment of the probe. The cofilin concentration-dependence of the phosphorescence intensity is sigmoidal, consistent with cooperative actin filament binding. Model-independent analysis of the anisotropies indicates that cofilin increases the rates of the microsecond rotational motions of actin. In contrast to the reduction in phosphorescence intensity, the changes in the rates of rotational motions display non-nearest-neighbor cooperative interactions and saturate at substoichiometric cofilin binding densities. Detailed analysis of the TPA decays indicates that cofilin decreases the torsional rigidity (C) of actin, increasing the thermally driven root-mean-square torsional angle between adjacent filament subunits from approximately 4 degrees (C = 2.30 x 10(-27) Nm2 radian(-1)) to approximately 17 degrees (C = 0.13 x 10(-27) Nm2 radian(-1)) at 25 degrees C. We favor a mechanism in which cofilin binding shifts the equilibrium between thermal ErIA-actin filament conformers, and facilitates two distinct structural changes in actin. One is local in nature, which affects the structure of actin's C terminus and is likely to mediate nearest-neighbor cooperative binding and filament severing. The second is a change in the internal dynamics of actin, which displays non-nearest-neighbor cooperativity and increases the torsional flexibility of filaments. The long-range effects of cofilin on the torsional dynamics of actin may accelerate P(i) release from filaments and modulate interactions with other regulatory actin filament binding proteins.

Actin Cytoskeleton↗

PubMed2005

Age-related decline in actomyosin function.

To understand the molecular basis of the functional decline in aging muscle, we examined the functional (actomyosin ATPase) and chemical (cysteine content) changes in actin and myosin purified from the muscles of young (4- to 12-month-old) and old (27- to 35-month-old) Fisher 344 rats. Using the soluble, catalytically active myosin fragment, heavy meromyosin (HMM), we determined the maximum rate (V(max)) and actin concentration at half V(max) (K(m)) of the actomyosin ATPase, using four combinations of actin and HMM from old and young rats. V(max) and K(m) were significantly lower when both actin and HMM were obtained from old rats than when both proteins were obtained from young rats. The number of reactive cysteines in HMM significantly decreased with age, but no change was detected in the number of reactive cysteines in actin. We conclude that aging results in chemical changes in myosin (probably oxidation of cysteines) that have inhibitory effects on the actin-activated myosin ATPase.

Actins↗

PubMed2004

Structural dynamics of actin during active interaction with myosin: different effects of weakly and strongly bound myosin heads.

We have used optical spectroscopy (transient phosphorescence anisotropy, TPA, and fluorescence resonance energy transfer, FRET) to detect the effects of weakly bound myosin S1 on actin during the actomyosin ATPase cycle. The changes in actin were reported by (a) a phosphorescent probe (ErIA) attached to Cys 374 and (b) a FRET donor-acceptor pair, IAEDANS attached to Cys 374 and a nucleotide analogue (TNPADP) in the nucleotide-binding cleft. Strong interactions were detected in the absence of ATP, and weak interactions were detected in the presence of ATP or its slowly hydrolyzed analogue ATP-gamma-S, under conditions where a significant fraction of weakly bound acto-S1 complex was present and the rate of nucleotide hydrolysis was low enough to enable steady-state measurements. The results show that actin in the weakly bound complex with S1 assumes a new structural state in which (a) the actin filament has microsecond rotational dynamics intermediate between that of free actin and the strongly bound complex and (b) S1-induced changes are not propagated along the actin filament, in contrast to the highly cooperative changes due to the strongly bound complex. We propose that the transition on the acto-myosin interface from weak to strong binding is accompanied by transitions in the structural dynamics of actin parallel to transitions in the dynamics of interacting myosin heads.

Actins↗

PubMed2002

Changes in actin and myosin structural dynamics due to their weak and strong interactions.

Figure 3 summarizes the effects of actomyosin binding on the internal and global dynamics of either protein, as discussed in this chapter. These effects depend primarily on the strength of the interaction; which in turn depends on the state of the nucleotide at the myosin active site. When either no nucleotide or ADP is bound, the interaction is strong and the effect on each protein is maximal. When the nucleotide is ATP or ADP.Pi, or the equivalent nonhydrolyzable analogs, the interaction is weak and the effect on molecular dynamics of each protein is minimal. The weaker effects in weak-binding states are not simply the reflection of lower occupancy of binding sites--the molecular models in Fig. 3 illustrate the effects of the formation of the ternary complex, after correction for the free actin and myosin in the system. Thus EPR on myosin (Berger and Thomas 1991; Thomas et al. 1995) and pyrene fluorescence studies on actin (Geeves 1991) have shown that the formation of a ternary complex has a negligible effect on the internal dynamics of both [figure: see text] proteins (left side of Fig. 3, white arrows). As shown by both EPR (Baker et al. 1998; Roopnarine et al. 1998) and phosphorescence (Ramachandran and Thomas 1999), both domains of myosin are dynamically disordered in weak-binding states, and this is essentially unaffected by the formation of the ternary complex (left side of Fig. 3, indicated by disordered myosin domains). The only substantial effect of the formation of the weak interaction that has been reported is the EPR-detected (Ostap and Thomas 1991) restriction of the global dynamics of actin upon weak myosin binding (left column of Fig. 3, gray arrow). The effects of strong actomyosin formation are much more dramatic. While substantial rotational dynamics, both internal and global, exist in both myosin and actin in the presence of ADP or the absence of nucleotides, spin label EPR, pyrene fluorescence, and phosphorescence all show dramatic restrictions in these motions upon formation of the strong ternary complex (right column of Fig. 3). One implication of this is that the weak-to-strong transition is accompanied by a disorder-to-order transition in both actin and myosin, and this is itself an excellent candidate for the structural change that produces force (Thomas et al. 1995). Another clear implication is that the crystal structures obtained for isolated myosin and actin are not likely to be reliable representations of structures that exist in ternary complexes of these proteins (Rayment et al. 1993a and 1993b; Dominguez et al. 1998; Houdusse et al. 1999). This is clearly true of the strong-binding states, since the spectroscopic studies indicate consistently that substantial changes occur in both proteins upon strong complex formation. For the weak complexes, the problem is not that complex formation induces large structural changes, but that the structures themselves are dynamically disordered. This is probably why so many different structures have been obtained for myosin S1 with nucleotides bound--each crystal is selecting one of the many different substates represented by the dynamic ensemble. Finally, there is the problem that the structures of actomyosin complexes are probably influenced strongly by their mechanical coupling to muscle protein lattice (Baker at al. 2000). Thus, even if co-crystals of actin and myosin are obtained in the future, an accurate description of the structural changes involved in force generation will require further experiments using site-directed spectroscopic probes of both actin and myosin, in order to detect the structural dynamics of these ternary complexes under physiological conditions.

Actins↗