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P M Curmi

Publications and source records attributed to P M Curmi.

28 records · Page 2Linked to original sources
PubMed1991

Mechanism of force generation studied by neutron scattering.

Neutron scattering has been used to compare the structure of myosin S1 that is free in solution to that when it is bound to F-actin. To achieve this, deuterated actin was obtained from D. discoideum that had been fed deuterated E. coli. This deuterated actin was rendered "invisible" to neutrons when dissolved in 94% D2O. The neutron scattering patterns obtained from S1 bound to deuterated actin were identical to those of free S1 except for oscillations due to S1's bound to the same actin filament. At low S1 to actin stoichiometries, these oscillations diminish and the patterns become indistinguishable. The apparent radius of gyration of S1 bound to actin is identical to that of free S1 when the stoichiometry is low. Thus, no changes in the structure of S1 were observed to a resolution of 2.5 nm. Computer modelling studies were used to evaluate the compatibility of models for the mechanism of force generation with the neutron data. These studies show that for powerstrokes greater than 5.0 nm, the data are consistent with more than 80% of the crossbridge maintaining a rigid conformation during force generation.

Actins↗
PubMed1991

Recent neutron scattering studies of muscle contraction and its control.

We have presented two applications of the method of neutron scattering utilizing selective deuteration of actin. In these experiments the actin was rendered effectively invisible to neutrons by matching the scattering-length densities of deuterated actin and the solvent. The scattering of neutrons by myosin S1 and by Tm bound to this actin was studied. For free chymotrypsin-generated S1 it was found that Rg = 4.0 +/- 0.15 nm, while for papain-generated S1 it was found that Rg = 4.6 +/- 0.2 nm. Upon binding of papain-generated S1 to actin at low NS1/N actin ratios, the change in Rg in difference experiments was delta Rg = 0.05 +/- 0.15 nm. This lack of significant change in Rg in the very low-s domain confirms and extends our earlier neutron scattering work in the higher-s domain. The longest chords of S1, as well as shorter ones, are not significantly altered upon actin binding. These results indicate that muscle contraction does not occur as a result of large-scale changes in S1 structure. In actin-Tm complexes, a measurement of the mean cross-helix separation, d, of Tm molecules has been made using neutron scattering. With deuterated actin matched out in 93% D2O buffer, it was found that d = 7.9 +/- 0.3 nm. This value is in good agreement with a model based on Tm crystallography and also with recent electron microscopy results. These experiments demonstrate the feasibility and value of neutron diffraction and scattering techniques in the study of muscle contraction and its control. One can expect that the further employment of emerging cell biology techniques for generating deuterated proteins will aid our understanding of muscle in the future.

Actins↗
PubMed1988

Comparison of the structure of myosin subfragment 1 bound to actin and free in solution. A neutron scattering study using actin made "invisible" by deuteration.

The structure of subfragment 1 (S1) bound to F-actin has been compared to the structure of free S1 using neutron scattering. The F-actin was rendered "invisible" to neutrons by selective deuteration and solvent contrast matching. Highly deuterated actin was purified from the slime mold Dictyostelium discoideum, which was fed deuterated Escherichia coli. The properties of this actin were found to be similar to those of protonated actin. The neutron-scattering pattern of S1 bound to this "invisible" actin was compared to that of free S1. At near-physiological ionic strength, a strong interference effect was observed, which arose from pairs of S1 molecules cross-linking actin filaments. However, at low ionic strength the only differences that could be observed were attributed to interference effects between neutrons scattered from S1s bound randomly to equivalent sites on an actin filament. These effects became negligible as the fraction of actin sites occupied by S1 approached zero. Thus, we conclude that the scattering by S1 attached to F-actin is identical with that of free S1, to a resolution of about 2.5 nm. The difference in apparent radii of gyration is less than 0.05 nm. Modeling calculations have been carried out to determine the sensitivity of neutron scattering to possible S1 deformations. The calculations showed that deformations of the structure of S1 that are large enough ultimately to produce a powerstroke of 5 nm or greater are only consistent with the data if they involve at most about 20% of the S1 mass. These results restrict the class of plausible models describing force generation in muscle contraction.

Actins↗
PubMed1988

Tertiary structure of plant RuBisCO: domains and their contacts.

The three-dimensional structure of ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO), has been determined at 2.6 A resolution. This enzyme initiates photosynthesis by combining carbon dioxide with ribulose bisphosphate to form two molecules of 3-phosphoglycerate. In plants, RuBisCO is built from eight large (L) and eight small (S) polypeptide chains, or subunits. Both S chains and the NH2-terminal domain (N) of L are antiparallel beta, "open-face-sandwich" domains with four-stranded beta sheets and flanking alpha helices. The main domain (B) of L is an alpha/beta barrel containing most of the catalytic residues. The active site is in a pocket at the opening of the barrel that is partly covered by the N domain of a neighboring L chain. The domain contacts of the molecule and its conserved residues are discussed in terms of this structure.

Amino Acid Sequence↗
PubMed1984

Actin tube formation: effects of variations in commonly used solvent conditions.

Crystalline tubular aggregates of actin spontaneously assemble in the presence of certain of the lanthanide ions. These tubes are now known to contain a high degree of structural order and it has been suggested that they may be sensitive to small changes in the primary sequence. However, there have been no detailed studies of the effects of solution conditions associated with their formation. In this report we systematically examine the effects of lanthanide ion concentration, ionic radius, adenosine nucleotide concentration, divalent cation concentration, pH, KCl concentration and incubation time. The stringent control of these parameters leads to a high degree of predictability of the structural parameters of the tubes and will thus be of use in identifying actin isozymes.

Actins↗
PubMed1983

A re-investigation of actin monomer conformation under polymerizing conditions based on rates of enzymatic digestion and ultraviolet difference spectroscopy.

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.

Actins↗
PubMed1982

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.

Actins↗
PubMed1982

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.

Actins↗
PubMed1981

Crystalline actin tubes. III. The interaction of scandium and yttrium with skeletal muscle actin.

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.

Actins↗