Structural and functional domains on actin.
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Biomedical subjects
C G dos Remedios
Publications and source records attributed to C G dos Remedios.
Differential co-expression of alpha-actin genes within the human heart.
Human cardiac muscle has been studied to determine whether the ratio of cardiac alpha-actin to skeletal alpha-actin varies between the different chambers of the human heart taken from a single individual. Using mRNA dot-blots, and DNA probes specific for the cardiac and skeletal alpha-actin isotypes, we have found that both cardiac and skeletal alpha-actin mRNAs are present and co-expressed throughout the human heart. The pattern of alpha-actin co-expression in the left and right ventricles and in the interventricular septum is approximately the same, with cardiac alpha-actin being the dominant isotype (approx. 80% of total). However, the left atrium has a different relative composition of the two actins, with an even higher level of cardiac alpha-actin expression (95% of total).
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Fluorescence energy transfer between Cys-10 residues in F-actin filaments.
Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-[2-((iodoacetyl)amino)-ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) as a fluorescence energy donor and 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) as the acceptor. Both labels were covalently attached to Cys-10 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Cys-10 was calculated to be 23 A. This corresponds to a distance between adjacent sites along the long pitch helix of 56.1 A and along the genetic helix of 53.3 A.
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The distance separating Cys-10 from the high-affinity metal binding site in actin.
The fluorescence resonance energy transfer between 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) attached to Cys-10 residue and Co2+ bound to the high affinity metal site was measured. The resonance energy transfer efficiency was 8 +/- 2%, which corresponds to a distance of 2.1 nm using the absorption spectrum of Co-EDTA and 3.0 nm using the more relevant absorption spectrum of Co2+ bound to carboxypeptidase as a model spectrum of Co2+ bound to actin, respectively.
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Selective labelling of Cys-10 on actin.
Conditions are described for the selective modification of Cys-10 on actin achieved following the blocking of the more reactive Cys-374. Labelling of Cys-10 did not affect the formation of actin filaments. This residue should be capable of serving as a site for fluorescence donors or acceptors and thus will be a useful locus to probe F-actin structure.
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Conformational changes in actin resulting from Ca2+/Mg2+ exchange as detected by proton NMR spectroscopy.
Skeletal muscle actin can be maintained in a monomeric form in very low ionic strength solutions as well as in high concentrations (0.6 M) of MgCl2 or CaCl2. 400-MHz 1H-NMR spectra revealed characteristic changes which show that the conformation of actin alters by exchanging Ca2+ for Mg2+ in the single high-affinity cation binding site. When all low-affinity cation binding sites are filled (in the presence of high concentrations of Ca2+ or Mg2+), the spectra show that actin conformation differs from that in low-ionic-strength buffer. A comparison of actin in 0.6 M CaCl2 and 0.6 M MgCl2 revealed that the environment of only a small number of protons is affected by the exchange. A new proposal for the essential steps involved in actin polymerization is presented.
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The environment of the high-affinity cation binding site on actin and the separation between cation and ATP sites as revealed by proton NMR and fluorescence spectroscopy.
The lanthanide ions Lu3+ (diamagnetic) and Gd3+ (paramagnetic broadening probe) were used to displace Ca2+ from the high-affinity cation binding site on G-actin. The effects of these higher-affinity ions on the proton nuclear magnetic resonance spectrum of actin were recorded. The aliphatic proton envelope in the Gd-actin sample exhibited a complex array of changes due to the proximity of Gd to several aliphatic residues. No such changes were observed in the diamagnetic Lu-actin control spectrum. By contrast, the aromatic proton envelope remained largely unaffected in both Gd-actin and Lu-actin samples. However, the adenosine moiety on the actin-bound ATP became increasingly mobilized without the triphosphate chain being released from the ATP binding site. Maximum adenosine mobilization occurred with approximately 1 mol of lanthanide ion bound per mol of actin. The absence of changes in the aromatic proton envelope suggests that the high-affinity cation binding site is in a region well removed from the adenosine moiety of bound ATP as well as any aromatic side-chains. The separation of the ATP and cation sites was further explored using the fluorescent ATP analogues FTP and epsilon-ATP. Tb3+ bound to the high-affinity cation site was found to be separated by 16 A from the FTP chromophore bound to the nucleotide binding site on actin. Since this distance is greater than can be accommodated on a model of the Tb-ATP complex, we conclude that the sites are physically separate. This conclusion was further reinforced by experiments involving the quenching of epsilon-ATP fluorescence by Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)
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Monoclonal antibodies directed against skeletal muscle actin.
The development of a monoclonal antibody directed against rabbit skeletal muscle monomeric actin is described. The production of the monoclonal antibody followed a standard hybridoma technique, the antibody being purified by affinity chromatography. It was found to be of the IgM class. Antibody specificity for rabbit skeletal actin was demonstrated by radioimmunoassay. The antibody failed to bind to actin in Western Blot experiments, presumably due to modification of the antigenic determinant on actin during the Western Blot procedure. The antibody was also shown to bind to two other isotypes of actin, i.e. actin from squid mantle muscle and bovine myocardium.
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Crystalline actin tubes. V. The effect of Th4+ on actin and the role of ionic charge in tube formation.
NMR and UV spectroscopy reveal that the tetravalent thorium ion interacts with actin in an identical fashion to the trivalent lanthanide ions. UV spectra and viscometry data suggest that the actin-bound Th4+ has an ionic radius of about 110 pm. Th4+ also contributes to the formation of crystalline actin tubes under appropriate conditions. The factors governing whether tubes form appear to be cationic radius and total charge on the actin monomer, rather than the valency of the ion per se.
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Conformational studies of G-actin containing bound lanthanide.
The presence of bound Ln ions gives rise to changes in the ultraviolet absorbance of G-actin. Previously, Ln ions have been shown to mobilize the adenosine moiety of ATP bound to the actin monomer. The changes in ultraviolet absorbance appear to be fully attributable to this mobilization. Any structural changes induced in G-actin by the substitution of Ln ions for Ca2+ must be small enough so as not to perturb the environment of any aromatic chromophores other than those exposed by the mobility of the nucleotide. This conclusion is supported by measurements of the rates of proteolytic digestion of actin with and without bound Ln ions. The effect of Ln ions on the actin-bound nucleotide is not simply due to the increase in positive charge on the actin monomer. This is demonstrated by the absence of any ultraviolet spectral changes on the addition of a fivefold excess of Ca2+. Analysis of the ultraviolet difference spectrum obtained when Ln ions bind to G-actin indicates that at least one tryptophan residue is partially exposed to the solvent coincident with mobilization of the nucleotide. Tb3+ luminescence enhancement studies indicate that there are no Trp, Tyr or Phe residues in the vicinity of the high-affinity cation site. These results suggest that the exposed Trp may be close to the nucleotide.
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Differences between cardiac and skeletal muscle actins.
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Crystalline actin tubes. IV. Structural information on actin monomers obtained from computer-averaged lattice images.
Crystalline actin tubes can be formed in the presence of Pr(III) and examples can be selected which are opened along their length. The structural unit cell seen in electron micrographs were computer-averaged to obtain the projected size and shape of the actin monomer. There are two monomers per unit cell, each related by a two-fold axis perpendicular to the plane of the projection. Each 3.3 x 5.5 nm monomer was markedly asymmetric. Skeletal muscle actin strongly resembles Acanthamoeba actin (Aebi et al. (1980) Nature 288, 296) although the projected dimensions may vary slightly.
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Proton nuclear magnetic resonance and electron paramagnetic resonance studies on skeletal muscle actin indicate that the metal and nucleotide binding sites are separate.
The distance separating the high-affinity binding sites of actin for a divalent metal ion and nucleotide was evaluated by using high-resolution proton NMR and EPR spectroscopy. Replacement of the Ca2+ or Mg2+ bound to the high-affinity divalent cation site of G-actin by trivalent lanthanide ions such as La3+, EU3+, or Gd3+ results in an increase in the mobility of the bound ATP as observed in the NMR spectra of G-actin monomers. Little difference was observed between the spectra obtained in the presence of the diamagnetic La3+ control and the paramagnetic ions Eu3+ and Gd3+ which respectively shift and broaden the proton resonances of amino acids in the vicinity of the binding site. Analysis of the NMR spectra indicates that the metal and nucleotide binding sites are separated by a distance of at least 16 A. In the past, the metal and ATP have been widely assumed to bind as a complex. Further verification that the two sites on actin are physically separated was obtained by using an ATP analogue with a nitroxide spin-label bound at the 6' position of the purine ring. An estimate of the distance was made between the site containing the ATP analogue and the paramagnetic ion, Mn2+, bound to the cation binding site. These EPR experiments were not affected by the state of polymerization of the actin. The data obtained by using this technique support the conclusion stated above, namely, that the cation and nucleotide sites on either G- or F-actin are well separated.
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Crystalline actin tubes. I. Is the conformation of the lanthanide-induced actin tube monomer more like F-actin than G-actin?
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.
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Crystalline actin tubes. II. The effect of various lanthanide ions on actin tube formation.
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.
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Ionic radius specificity of cardiac muscle.
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Actin microcrystals and tubes formed in the presence of gadolinium ions.
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Is there a third type of filament in striated muscles?
Using glycerinated rabbit psoas myofibrils we demonstrate by electron microscopy that there is a filamentous material which remains after extraction of the fibrils with a modified Hasselbach-Schneider solution (containing 1.0 M KCl) followed by extraction with 0.6 M KI. The fibrils were visualized by conventional negative staining techniques using either 2% ammonium molybdate or 1% uranyl acetate. The appearance of the extracted fibrils together with their solubility properties strongly suggests that the extraction-resistant filamentous material is composed of connectin, an elastic protein recently characterized from muscle tissues.
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