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This review deals with the structure of the actin monomer, its assembly into filaments and the loci on F-actin involved in binding myosin. Two distinctly different arrangements of monomers have been suggested for actin filaments. One model proposed by Holmes et al. is well developed. It places the so-called 'large' domain close to the filament axis and the so-called 'small' domain out near the surface of the filament. A second, less-well developed, model proposed by Schutt et al. locates the 'small' domain close to the filament axis and they rotate the monomer so that 'bottom' of the 'large' domain is at the highest radius. We analyze the available evidence for the models of F-actin derived from X-ray diffraction, reconstructions from electron micrographs, fluorescence resonance energy transfer spectroscopy, chemical cross-linking, antibody probes, limited proteolysis, site-directed and natural mutations, nuclear magnetic resonance spectroscopy and other techniques. The result is an actin-centered view of the loci on actin which are probably involved in its interaction with the myosin 'head'. From these multiple contacts we speculate on the sequence of steps between the initial weak-binding state of S-1 to the actin filament through to the stable strong-binding state seen in the absence of free Mg-ATP, i.e., the rigor state.

The ability of skeletal muscle actin to aggregate in the form of crystalline tubular structures was examined using all 14 of the available trivalent lanthanide cations. Under conditions which normally cause F-actin formation, (0.1 M KCl, pH 6.9) only those lanthanide ions which interact with actin with a molar ratio of 5:1 (Ce3+, Pr3+, Nd3+, Sm3+ and Eu3+) or 6:1 (Gd3+, Tb3+, Dy3+ and Ho3+) can promote the formation of ordered tubular structures. Under the same conditions, the remaining ions (Er3+, Tm3+, Yb3+, Lu3+ as well as La3+) interact with a 7:1 molar ratio of lanthanide to actin, but these were unable to form actin tubes. Actin tube dimensions undergo systematic changes depending on which lanthanide ion binds. The dimensions of the actin monomers and their packing arrangement (i.e. number of rows of subunits per helical repeat and the pitch angle) determines the actin tube diameter. It is suggested that the failure of actin tube formation when 7 lanthanide ions bind is due to additional charge on the monomer, possibly conferring on it a net positive charge.

Intramonomer fluorescence resonance energy transfer between the donor epsilon-ATP bound to the nucleotide site and the acceptor N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) or 4-dimethylaminophenyl-azophenyl-4'-maleimide bound to Cys-10 in G-actin was measured. The donor-acceptor distance was calculated to be about 40 A. The intermonomer energy transfer in F-actin occurring between epsilon-ADP and DABMI was also measured. The radial coordinate of Cys-10 was calculated to be 25 A based on the helical symmetry of F-actin and the recently calculated radial coordinate of the nucleotide binding site in F-actin i.e. 25 A (Miki, M., Hambly, B. and dos Remedios, C.G. (1986) Biochim. Biophys. Acta 871, 137-141). (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Corresponding distances separating the donor nucleotide in one monomer from acceptors on Cys-10 in the first and second nearest neighbours in F-actin are 39-40 A and 41-43 A.

We varied the molar ratio of added lanthanide ion to skeletal muscle actin (M3+/A) and observed their effects on the change in reduced viscosity (Nred) in the presence of polymerizing quantities of salt (0.1 M KC1). Once the concentration of the lanthanide ion exceeds the concentration of the nucleotide present (0.2 mM ATP), we noted that with M3+/A ratios up to 4: (a) there was a sharp peak in the observed Nred above the level achieved by control F-actin; (b) the magnitude of (a) was shown to be a function of the initial G-actin concentration. With an M3+/A ratio of greater than 4 we observed: (i) a sharp fall in the observed Nred; (ii) the formation of an insoluble aggregate of actin; (iii) the formation of (ii) was completely reversed by removal of the M3+; (iv) a complete inhibition of the ATP hydrolysis which always accompanies the G- to F-actin transition; (v) the number of mol of M3+ required to completely inhibit the rise in Nred (above the viscosity of G-actin) was a function of the ionic radii of the 11 lanthanide ions tested; and (vi) the effects described in (i) were not mimicked when the initial protein was in the F form. In the absence of added KCI, divalent cations (e.g. Mg2+) polymerize G-actin but this effect is not mimicked by the addition of the lanthanide ions. However, under these conditions the lanthanide ions cause the formation of an insoluble aggregate of actin. We conclude that with greater than 4 mol of lanthanide ions, G-actin aggregates in a form which contains little or no F-actin and that the lanthanide ion-induced aggregates are therefore different from the Mg2+-induced F-actin paracrystals.

The structural relationship between F-actin filaments and the biologically active fragments of myosin (either as myosin subfragment-1 or heavy meromyosin) has been investigated using the technique of fluorescence energy transfer. Donor and acceptor probes were used to obtain the following inter- and intramolecular distances. Energy transfer was measured: (1) from the SH1 groups of the myosin 'heads' to the nucleotide sites of F-actin (in the absence of free nucleotide); (2) from the SH1 groups of myosin to multiple probes on the surface of the actin filament; (3) from the nucleotide-binding sites of F-actin to the ATPase sites of myosin; (4) from the ATPase sites of myosin to the nucleotide-binding sites of F-actin; (5) from the SH1 sites of myosin to the nucleotide-binding sites of F-actin; and (6) from the Cys-373 residues of F-actin to the nucleotide binding sites of F-actin. We observed very little energy transfer between the probes on actin and the probes on myosin (10% or less) and we observed a large transfer between the actin Cys-373 and the actin nucleotide. These data strongly suggest that both the SH1 moiety and the ATPase site of myosin are located more than 6 nm from the actin sites. When these distances are combined with similar measurements by other authors and inserted into the most recent three-dimensional reconstruction of electron micrographs of the acto-subfragment-1 complex, it is apparent that the SH1 and the ATPase sites on myosin are not located adjacent to actin and are most probably located in the half of the myosin head that is distal from actin in the actomyosin complex.

Fluorescence energy transfer between nucleotide binding sites in an F-actin filament was measured using 1-N6-ethenoadenosine diphosphate (epsilon-ADP) as a fluorescent donor and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) as an acceptor, both of which were bound to F-actin. Taking into consideration the helical structure of the F-actin filament, the radial coordinate of the nucleotide binding site was calculated to be 25 A, which corresponds to a distance between these sites along the long-pitch helix of 56.3 A and along the genetic helix of 56.7 A.

There have been several reports which describe a conformational change of G-actin monomer in the presence of 0.1 M KCl. This altered monomer has been variously named, depending on whether the authors believed that it resembles G-actin, F-actin or has a conformation of its own. In this report we re-examine the experimental evidence for these proposals. The techniques include measurements of the rates of proteolytic digestion as well as near and far ultraviolet difference spectroscopy of actin in the presence and absence of KCl. We conclude that there is no compelling evidence for proposing a novel form of actin monomer.

The effect of the trivalent cations scandium (Sc3+) and yttrium (Y3+) on the conformation of G-actin was examined using ultraviolet difference and high resolution 1H-NMR spectroscopy. A comparison was made with data obtained previously with the trivalent lanthanide cations (Ln3+). These results indicate that the first and subsequent Y ions (ionic radius 101.9 pm) behave exactly like Ln3+. Sc3+ is a smaller ion (87 pm) than any of the Ln3+. The first Sc3+ binds to a site on actin that is inaccessible to Mg2+, Y3+ and Ln3+. However, the second Sc3+ to bind behaves like an Ln3+. On replacing the native divalent cation (Mg2+), both Y3+ and Sc3+ mobilize the adenine ring of ATP bound to actin, thus exposing underlying residues to the solvent. When Y3+ and Sc3+ saturate their binding sites on actin, and when the ionic strength is raised to 0.1 M with KCl at pH 6.9, the actin aggregates. Y3+ binds to actin with a ratio of 6 : 1 and induces the aggregation of actin into crystalline actin tubes, whilst Sc3+ binds with a ratio of 8 : 1 and induces amorphous actin aggregates. These results are consistent with the suggestion that actin tubes are induced by trivalent cations, principally on the basis of their binding stoichiometry, which is determined by ionic radius.

Actin, isolated from rabbit skeletal muscle, forms highly-ordered aggregates when it binds six moles of the lanthanide ion, Gd3+. In the presence of 0.1 M KCl, these aggregates are referred to as actin tubes. The monomer contained in the repeating subunit of these tubes possess a number of functional characteristics which include: (i) binding to myosin or subfragment-1 of myosin; (ii) rapid conversion into filamentous Gd-actin which can activate myosin ATPase activity; (iii) a slow rate of exchange of the bound nucleotide; (iv) a slow rate of exchange of the metal cation; (v) a resistance to digestion by proteolytic enzymes. Additionally, the monomer of the Gd-actin tube structures appears to stoichiometrically bind ATP and exhibit a lower minimum protein concentration for tube formation than is needed for the formation of F-actin. The properties listed above suggest that the actin monomer, which comprises the Gd-actin tubes, bears little resemblance to either the G-actin monomer or the recently-described actin monomer conformation that exists under conditions that favour polymerization. The data suggest that the actin molecules which comprise the Gd-actin tube structures contain sites which bind myosin, nucleotide and metal cations and that these sites are similar to the sites on F-actin.

Skeletal muscle actin above a critical concentration polymerizes in physiological concentrations of KCl. Earlier studies have concluded that evidence exists for a monomeric species of actin with a conformation distinct from that of G-actin. Re-investigations of these earlier studies, however, have cast doubt on the concept of a new monomeric actin species. In this study we have adopted two methods, high-resolution proton nuclear magnetic resonance and near ultraviolet circular dichroism spectroscopy, to investigate the existence or otherwise of the putative monomer conformation variously called F-monomer, G-actin or KCl-monomer. For proton nuclear magnetic resonance spectroscopy, unmodified actin maintained below its critical concentration as well as higher concentrations of two chemically modified, unpolymerizable actin samples were studied in the absence and presence of KCl. No difference was found in the environment of even a single proton within the entire actin structure. For circular dichroism we studied actin maintained below its critical polymerization concentration and found very little change in ellipticities when KCl was added to the G-actin solution. We therefore conclude that there is no species of actin monomer with a conformation distinct from that of G-actin.