Biomedical subjects
Publications and source records attributed to R S Goody.
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Muscle contraction takes place by the cyclic interaction of two-stranded helical actin filaments with protuberances from the myosin filaments known as cross-bridges. The nature of the contractile process demands that the myosin cross-bridge be highly polymorphic. Biochemical evidence points to the cross-bridge having a domain structure such that contraction may be a manifestation of the rearrangement of these subunits. An essential step in contraction may be the initial binding of a small hinged domain of the cross-bridge (the 'nose-cone') to actin, followed by a series of changes ending in the formation of a second union (the rigor bond) with an actin monomer in the neighbouring strand of the actin helix.
Synchrotron radiation was used for low-angle X-ray diffraction to monitor structural changes produced in insect flight muscle during fixation, dehydration and embedding for electron microscopy of thin sections. Fibre bundles were fixed by cold glutaraldehyde in one of three states, namely rigor, ATP or AMPPNP, followed by additional cross-linking treatment. No heavy metals were used before embedding. During fixation-embedding, all specimens lost the continuous actin layer lines of spacing 11-5 nm, shrank 18-21% in lattice spacing, shrank 0.5-2.5% in axial spacings and showed equatorial intensity changes which were similar for all three states, while the well-sampled inner layer lines (39-13 nm) were preserved with different fidelity in each state, highest for rigor and lowest for ATP. In different AMPPNP bundles, these layer lines indicated different degrees of unexplained shift (from slight to total) towards the structure of muscle fixed in ATP. Fixation in ATP caused obvious gain of intensity on 39, 19 and 13 nm layer lines, which can be interpreted as trapping of myosin crossbridge attachments to actin; this artifact was unchanged by seven variations in fixation conditions. Fixation in rigor gave no indication of crossbridge detachment nor of the presence or alteration of any significant population of non-bridging myosin heads. X-ray monitoring allowed selection of best-preserved samples for subsequent electron microscopy. The rapid pattern-recording possible with synchrotron X-ray intensity allowed us to complete and compare experiments with many fibre bundles from a single glycerinated Lethocerus muscle.
This paper presents electron microscopy, supported by optical diffraction and filtering of images from 100 nm and 25 nm sections, to complement the companion report of X-ray diffraction monitoring (immediately preceding this article) performed on the same insect flight muscle specimens during fixation, dehydration and embedding. Glycerinated Lethocerus fibre bundles initially fixed in rigor, in ATP relaxing buffer, or in 1 mM AMPPNP at 2 degrees C, gave thin-section images from each state whose optical transforms match the distinctive X-ray diffraction patterns from the embedded samples. For rigor and relaxed states, this extends and confirms a long-known correlation between X-ray patterns and EM image regularities. For the AMPPNP state, such correlation is here fully developed for the first time, and involves a new and distinctive EM image pattern of the crossbridge array, clearly different from a previously reported structure in AMPPNP-treated muscles that appears identical to fixed relaxed muscle. We found this latter artifact of 'AMPPNP-relaxed structure' in many fibres from our best AMPPNP specimen, but could identify other fibres which retained the distinctive AMPPNP structure, known to be dominant in this specimen from the X-ray pattern. The true AMPPNP structure shows features of both the ATP-relaxed and rigor crossbridge patterns, not as separate patches, but hybridized uniformly along each filament and throughout each affected sarcomere and fibre. It presents a 14.5 nm repeat of striping and lateral projections along thick filaments, together with variously angled crossbridge attachments to actin that form a 38.7 nm repeat of diffuse chevrons or deltoids replacing the more clearly delinated rigor double chevrons. The associated optical transform has the typical AMPPNP features, that is, it has in common with rigor a strong 19.3 nm layer line and strong second to fourth row line sampling on the 38.7 nm layer line, it has in common with relaxed patterns a strong 14.5 nm meridional and layer line, but it uniquely shows no intensity at the first row line on the 38.7 nm layer line (the 10.3 X-ray reflection), where rigor and relaxed transforms always show high intensity. The processing artifacts which intensify the 10.3 reflection, and produce the weak 19.3 nm layer line (a gain of intensity for ATP but a loss for the AMPPNP state), throughout ATP specimens and in those analogue-treated fibres showing AMPPNP-relaxed structure, might indicate trapping and accumulation of minority populations within the native equilibrium distribution of crossbridge conformations in each nucleotide state.
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Equilibrium constants for the formation of a ternary complex between actin, myosin subfragment 1 (S1) and the non-hydrolyzable ATP analog adenosine 5'-[beta, gamma-imido]triphosphate (Ado PP[NH]P) were determined from light-scattering titrations under a variety of conditions. The affinities of S1 (binding constant K1) and acto . S1 (K4) for AdoPP[NH]P have relatively low dependencies on temperature (delta H degrees approximately equal to - 15 - 30 kJ mol-1) and ionic strength, in contrast to the affinities of S1 (K2) and S1 . AdoPP[NH]P (K3) for actin which are influenced quite strongly by temperature (delta H degrees approximately equal to 50 - 65 kJ mol-1) and ionic strength, K2 decreasing by a factor of 10 - 15 between I = 0.05 M and I = 0.2 M and K3 decreasing by a factor of 5.K1, and by detailed balance K2 as well, were found to be about 10-times higher than hitherto reported values (K1 = 3.4 X 10(7) M-1, K2 = 6 X 10(8) M-1, at 24 degrees C,I = 0.09 M, pH 8.0). The binding of ADP to S1 is about 10-fold weaker than that of AdoPP[NH]P, being however much more exothermic (delta H degrees = - 70 kJ mol-1 at I = 0.1 M) and having a negative standard entropy change (delta S = - 125 J mol-1 K-1), in contrast to AdoPP[NH]P binding for which the calculated delta S had positive values. The observed rate constant of dissociation of acto . S1 by AdoPP[NH]P showed an almost hyperbolic dependence on the nucleotide concentration, reaching a maximum of 15 s-1 at I = 0.055 M and 5 s-1 at I = 0.275 M, pH 8.0, 23 degrees C; at 5 degrees C this value was somewhat higher. The rate constant of dissociation of AdoPP[NH]P from its complex with acto . S1 was estimated to exceed 400 s-1 at 23 degrees C, and to be of the order of 150 s-1 at 4 degrees C. The observed rate constant for the association of the S1 . nucleotide complex and actin was proportional to actin concentrations up to 60 microM, thus defining an apparent second-order rate constant of 2 X 10(4) M-1 s-1 at I = 0.125 M and 23 degrees C. A reaction scheme is proposed in which isomerizations of the acto . S1 and acto . S1 . nucleotide complexes can occur.
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A composite data set in Fourier space has been produced from electron micrographs of negatively stained decorated actin and X-ray fibre diffraction patterns of glycerinated insect muscle "decorated" with rabbit myosin cross-bridges. It is shown that a simple model of actin and myosin may be adjusted to fit this data. The model conforms to the Taylor-Amos geometry for the acto-myosin interaction in accordance with the idea that the cross-bridge binds to two neighbouring actin monomers on different strands.
The structure of the MgGDP complex at the active site of elongation factor (EF-Tu) has been investigated by using phosphorothioate analogs of GDP in the absence and presence of various metal ions, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) measurements. The high stereoselectivity of EF-Tu for the diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDP[alpha S]) is independent of the nature of the metal ion and is caused by the interaction of the protein with the alpha-phosphate of GDP. By using GDP analogs where the oxygens at either the alpha-phosphate or the beta-phosphate have been selectively labelled with 17O and measuring their effect on the EPR spectrum of EF-Tu-bound manganese we are able to show that only the beta-phosphate of GDP is coordinated to the metal ion in the EF-Tu . Me2+ . GDP complex. 31P-NMR studies on GDP and guanosine 5'-O-(2-thiodiphosphate) (GDP[beta S]) bound to EF-Tu indicate that in the EF-Tu . Me2+ . GDP complex Mg2+ interacts more strongly with the beta-phosphate than with the alpha-phosphate. Together with binding studies using GDP[beta S] our NMR results also indicate that the protein is complexed to the beta-phosphorous of GDP via two oxygens.
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The five possible analogues of ATP and the three possible analogues of ADP which contain single non-bridging sulphur atoms instead of oxygen in the polyphosphate structure have been used as probes of the interaction of nucleotides with myosin and actomyosin. Evidence is presented for the requirement of an alpha, beta, gamma-tridentate complex of magnesium and ATP as the substrate for myosin. Of the four possible tridentate MgATP diastereomers, the delta exo isomer (nomenclature of Cornelius & Cleland, 1978) appears to be the actual substrate.
The rate of exchange of actin-bound nucleotide is decreased by a factor of about 20 when actin is complexed with DNAase I without affecting the binding constant of calcium for actin. Binding constants of DNAase I to monomeric and filamentous actin were determined to be 5 X 10(8) M-1 and 1.2 X 10(4) M-1 respectively. The depolymerisation of F-actin by DNAase I appears to be due to a shift in the G-F equilibrium of actin by DNAase I. Inhibition of the DNA-degrading activity of DNAase I by G-actin is of the partially competitive type.
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