Biomedical subjects

K J Poole

Publications and source records attributed to K J Poole.

5 recordsLinked to original sources

PubMed1998

X-ray diffraction indicates that active cross-bridges bind to actin target zones in insect flight muscle.

We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. As active tension rose, the 14.5- and 7.2-nm meridionals fell, the first row line dropped at the 38.7 nm layer line while gaining a new peak at 19.3 nm, and three outer peaks on the 38.7-nm layer line rose. The first row line changes suggest restricted binding of active myosin heads to the helically preferred region in each actin target zone, where, in rigor, two-headed lead bridges bind, midway between troponin bulges that repeat every 38.7 nm. Halving this troponin repeat by binding of single active heads explains the intensity rise at 19.3 nm being coupled to a loss at 38.7 nm. The meridional changes signal movement of at least 30% of all myosin heads away from their axially ordered positions on the myosin helix. The 38.7- and 19.3-nm layer line changes signal stereoselective attachment of 7-23% of the myosin heads to the actin helix, although with too little ordering at 6-nm resolution to affect the 5.9-nm actin layer line. We conclude that stretch-activated tension of IFM is produced by cross-bridges that bind to rigor's lead-bridge target zones, comprising < or = 1/3 of the 75-80% that attach in rigor.

Actins↗

PubMed1995

An atomic model of the unregulated thin filament obtained by X-ray fiber diffraction on oriented actin-tropomyosin gels.

We present a model of the actin-tropomyosin complex in which the radial and azimuthal position of tropomyosin was adjusted to fit the X-ray fiber diffraction patterns from oriented actin-tropomyosin gels at a resolution of 1/8 A-1. We used the recently published atomic F-actin model for the calculations. The atomic model of tropomyosin was obtained by model-building a coiled coiled-coil structure from the tropomyosin sequence. The resulting atomic model is strongly preferred and shows strong electrostatic interactions between charged side-chains of tropomyosin residues and actin residues in subdomain 3 and subdomain 4. Furthermore, calculations of enthalpies based upon electrostatic interactions indicate that there is a favored rotational position of the tropomyosin core at the calculated azimuthal and radial position given by the X-ray refinement. Rotations of the tropomyosin strand out of this position turn strongly attractive electrostatic interactions into repulsive forces. The resulting binding radius of 39 A and the determined azimuthal position of tropomyosin are in good agreement with electron microscopy reconstructions and neutron diffraction experiments. Furthermore, the calculated position of tropomyosin would still partly block the rigor interaction of myosin cross-bridges with actin, whereas it very likely allows undisturbed binding of the cross-bridges in a weak binding state.

Actins↗

PubMed1991

Time-resolved X-ray diffraction studies on stretch-activated insect flight muscle.

The specific feature of stretch activation of the indirect flight muscle of the tropical waterbug Lethocerus was used to correlate mechanical and structural aspects of muscle contraction. The time courses of the changes in intensities of the strongest equatorial reflections, the (10) and (20) and of the first meridional reflection at 14.5 nm-1 were monitored using synchrotron radiation as a high intensity X-ray source. The ratio of the intensities of the equatorial reflections, (I20/I10), which reflects the mass distribution within the filament lattice array, increases by about 10% relative to the Ca(2+)-activated level when a rapid stretch is imposed, compared with a 200% change seen when fibres change from the relaxed to the rigor state, while the spacing of the lattice planes decreases by about 1%. The intensity of the first meridional reflection at 14.5 nm-1 decreases by about 35% during stretch activation with a slightly faster time course than the delayed tension increase. The results suggest that the average structure of cycling crossbridges is different from that present in the rigor state.

Actin Cytoskeleton↗

PubMed1991

Dynamic X-ray diffraction measurements following photolytic relaxation and activation of skinned rabbit psoas fibres.

1) The ATP binding and crossbridge dissociation in muscle fibres is as fast as in solution, has a Q10 ca. 2-3, and is not measurably strain sensitive. 2) The final ADP release from the AM.ADP state achieved by adding ADP to rigor fibres must be greater than or equal to 69 sec-1 at 10 degrees C, and the combination of this rate and the ADP rebinding rate at 1 mM ADP limits the ATP induced crossbridge dissociation rate at greater than 2 mM ATP, but these kinetics were not strain sensitive. The strain sensitive steps must occur earlier on the attached pathway. 3) On activation, the equatorial changes thought to reflect crossbridge attachment are faster than tension production. The 10 intensity may change slightly ahead of the 11. This rate was not very temperature sensitive unlike the tension producing step in the mechanism. 4) The re-equilibration of equatorial intensity levels was much faster on activation from the rigor state than from the relaxed state. We conclude that crossbridges do not necessarily move far from the thin filaments when they detach in a fully activated thin filament system. 5) The 14.3 nm meridional intensity increases greater than 200% on fibre activation at 24 degrees C. The structural reorganisation of the heads responsible for this increase is associated with the tension generating step in the ATPase mechanism rather than the initial binding of bridges.

Adenosine Diphosphate↗

PubMed1988

The time course of changes in the equatorial diffraction patterns from different muscle types on photolysis of caged-ATP.

Using the synchrotron X-ray source at DESY, Hamburg, we have measured the time courses of changes in the strongest equatorial reflections from small bundles of chemically skinned fibres from insect flight muscle, Limulus muscle and rabbit psoas and soleus muscles following the photolytic release of ATP. In all preparations the release of ca. 2 mM ATP caused the tension to relax with a complex time course, the final relaxation rates in insect and rabbit psoas fibres being ca. 10 times faster than those measured in rabbit soleus and Limulus fibres (ca. 50 ms cf. ca. 500 ms half times). However, in all fibre types there was a very rapid change in equatorial intensities towards relaxed values (half time less than 5 ms), the extent of this change and the occurrence of a slower phase of intensity change being dependent on the preparation. In insect the 1.0 and 2.0 intensities change rapidly to their relaxed values; in Limulus the 1.0 and 1.1 intensities change rapidly to within ca. 10% of their relaxed values; in rabbit psoas the initial, rapid 1.1 intensity fall is to within 30-40% of its relaxed value and is followed by a slower fall which appears to correlate with the rate of the final tension relaxation, e.g. phosphate ions accelerate both rates; in rabbit soleus the equatorial response is very similar to that of psoas fibres. These results are discussed in terms of the model of Goldman et al. in which a rapid ATP induced dissociation of rigor crossbridges is followed by the transient cooperative reattachment of some bridges which may proceed through at least part of the cross-bridge cycle.

Actomyosin↗