Yuichiro Maéda

Rabbit skeletal muscle myosin unfolded carboxyl-terminus and its role in molecular assembly

We have expressed in E. coli segments of the rod portion of rabbit skeletal fast muscle myosin and compared physical properties of two different species, LMM‐30 and LMM‐30C′. LMM‐30 consists of 263 amino acids including the original C‐terminus of myosin heavy chain. LMM‐30C′ is colinear with LMM‐30, but is devoid of 17 residues at the C‐terminus. 1H NMR spectroscopy indicates that the C‐terminus of LMM‐30, but not of LMM30C′ is unfolded and freely mobile. Furthermore, the present results show that the unfolded C‐terminus is essential for molecular assembly of LMM‐30; at pH 8.0 LMM‐30, but not LMM‐30C′, formed aggregates upon decreasing the ionic strength.

Time-Resolved Structural Studies on Insect Flight Muscle after Photolysis of Caged-ATP.

The time course of structural changes occurring on ATP-induced relaxation of glycerinated insect flight muscle from the rigor state has been investigated using synchrotron radiation as a source of high intensity x rays and photolysis of caged-ATP to produce a rapid rise in ATP concentration. Temporal resolutions of 1 ms for the strongest equatorial reflections and 5 ms for the 14.5 nm meridional reflection are attainable from single events (i.e., without averaging over several cycles). The equatorial intensity changes completely, the meridional intensity partially, towards their respective relaxed values on a much faster time scale than relaxation of tension. The results suggest that actively cycling bridges present shortly after ATP-release are either too few in number to be detected in the equatorial diffraction pattern or that their structure is different from that of rigor bridges.

Time-resolved x-ray diffraction study of the troponin-associated reflexions from the frog muscle.

The vertebrate skeletal muscle gives rise to a series of x-ray reflexions indexed as orders (n) of 77 nm, the even orders being meridional whereas the odd orders being near-meridional. The diffraction intensities associated with these reflexions originate from the axial period of 39 nm attributable to the repeat of troponin-tropomyosin on the thin filament. In the present study, the x-ray intensities of the furthest inner reflexions, A2 (n = 2) reflexion at an axial spacing of 1/39 nm-1 and A4 (n = 4) reflexion at 1/19 nm, of this series were measured with a time resolved manner. Upon activation of the frog striated muscle, the two reflexions underwent biphasic time courses of the intensity changes. With A2 reflexion, a rapid intensity increase by 16%, being completed by the time when tension rises to 5%, was followed by a slow intensity decrease down to 50%, which was associated with the tension rise. In both phases, lateral widths remained unchanged. A4 reflexion also behaves in the same way, although the first phase (the intensity increase) was not clear due to unsatisfactory statistics. We interpret phase one as being caused by conformational change of the troponin-tropomyosin complex upon binding of Ca2+ to troponin, whereas phase two being due to direct contribution of the mass of the myosin heads bound to the thin filament, although possible contribution of conformational changes of the regulatory proteins to phase two is not excluded. The results indicated that the calcium activation of the thin filament leads the onset of the actomyosin interaction.

X-ray diffraction studies on muscle regulation

Using the intensity of the outer part of the second actin layer line as an indicator of thin filament conformation in vertebrate muscle we were able to identify the four different states of rest, and the three states induced by the presence of Ca2+ ions, rigor bridge attachment and actively cycling bridges, respectively. These findings are in qualitative agreement with a number of biochemical studies by Eisenberg and Greene and others, indicating that activation of the thin filament depends both on Ca2+ ions and crossbridge binding. Yet quantitatively, the biochemical data and our structural data are contradictory. Whereas the biochemical studies suggest a strong coupling between structural changes of the thin filament and the ATPase activity, the structural studies indicate that this is not necessarily the case. Troponin molecules also change their conformation upon activation depending on both Ca2+ ions and crossbridge binding as demonstrated by the early part of the time course of the thin filament meridional reflections in contracting frog muscle. Low ionic strength which has been shown by Brenner and collaborators to increase weakly binding crossbridges in relaxed rabbit psoas muscle does not influence the intensity of the second actin layer line in this muscle. Yet in contracting frog muscle the increase of the second actin layer line increases very rapidly in one step, suggesting that weak binding bridges which are attached to actin prior to force production may indeed influence the thin filament conformation. It therefore appears that weakly bound bridges in the low ionic strength state do not have the same effect on the thin filament conformation as weakly bound bridges in an actively contracting muscle. Arthropod muscles like the thin filament regulated lobster muscles differ from vertebrate muscle in not showing an increase of the second layer line during contraction, which may have to do with differences in crossbridge attachment. The myosin-regulated molluscan muscle ABRM shows a large increase on the second actin layer line upon phasic contraction and a much smaller increase in catch or rigor, indicating that actively cycling bridges influence the thin filament conformation differently than catch or rigor bridges. Several pieces of evidence which we have briefly outlined in this paper suggest that the thin filament conformational changes we have observed do not arise solely from tropomyosin movements and that conformational changes of actin domains should be considered.

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.