Photon Factory Activity Report 2014 #32 (2015) B

BL-6A/201３G212

Structural change of myosin head in skeletal muscle fiber
without thin filament
Shigeru Takemori1*, Maki Yamaguchi1, Naoya Nakahara1, Masako Kimura2,Tetsuo Ohno1, Hideki Yamauchi1, Takayuki Suzuki 1, Toru Kurihara1
1) The Jikei University School of Medicine, 105-8461, Japan
2) Kagawa Education Institute of Nutrition, 350-0288, Japan
1. Introduction
In relaxed muscle, diffraction patterngives information about myosin structure.
However, when actin and myosin
undergo normal contractile interaction orpotentiatedcontractileinteraction
facilitated by phosphorylation of myosin
light chain, structural information ofmyosin
wasdissipatedduetointervention of actin structure. The aimof this experiment was elucidating
structural change of myosin itself during
ATP hydrolysis without intervention ofactin-myosin interaction to reveal
elementary process of chemomechanicalcoupling on myosin realizing muscle
contraction.
M → M-ADP-Pi → M-ADP → M
Scheme 1 Proposed ATP hydrolysis cycle on myosin
M:myosin without nucleotide,
M-ADP-Pi: ADP-Pi-bound myosin
M-ADP:ADP-bound myosin

2. Experiments
Skinned skeletal muscle specimens wereobtained by treatment of bundles
consisting of 3 skeletal muscle fibers
from rabbit psoas muscle with 0.5%
triton-X to destroy cell membrane.
Skinned skeletal muscle specimens werethen treated with 3 mg/ml gelsolin
purified from bovine serum [1] for 5
hours to remove actin filament from thespecimen. X-ray diffraction patterns inrelaxing (myosin is mainly in M-ADP-Pistate), ADP-rigor (myosin is in M-ADPstate), rigor (myosin is in M state) wereobtained using imaging plate system at20 degrees of Celsius. To remove
remnant ATP within the specimen,
hexokinase plus glucose system or
apyrase were used. P1,P5-di(adenosine-5’)
pentaphosphate (AP5A) was added toADP-rigor solution to inhibit myokinase
activity [2].
3. Results and Discussion

Myofilament spacing of thin-filamentextracted fibers
By extraction of thin filament, spacing
between thick filament reduced by 5nm
in relaxing condition. No difference wasdetected among relaxing, ADP-rigor, andrigor conditions.

Change of myosin layer line by thinfilament extractionBy extraction of thin filament, peak
position of myosin layer line in relaxing
condition did not change by extraction ofthin filament significantly (Fig.1: blue;
before extraction, black; after extraction).

Photon Factory Activity Report 2014 #32 (2015) B

Change of myosin layer line by transition
from M-ADP-Pi to M-ADP state
On ADP condition, peak height of myosin
layer line decreased and the peak
position slightly shifted toward meridiancompared with relaxing condition (MADP-Pi state).
It indicated that myosin head slightly
protruded from backbone and gained
increased mobility with transition fromM-ADP-Pi state to M-ADP state (Fig.2:
blue; M-ADP-Pi, green; M-ADP).

4. Conclusion
1. Myosin head was found to undergo
radial movement involved in ATPhydrolysis reaction even without actin.
2. Myosin head further shifted fromthick-filament backbone to actin bytransition from M-ADP state to M state.
3. This result provide fundamental
information for the understanding ofstructural change of myosin in normal or
potentiated contraction by myosin lightchain phosphorylation.

Fig.4
Change of myosin layer line by transition
from M-ADP to M state
In rigor state (myosin is in M state), peak
position of myosin slightly shifted toward
meridian compared with ADP condition（M-ADP state ). It indicated that myosin
heads slightly protruded from backbone
with transition from M-ADP to M (Fig.3:green; M-ADP, magenta; M).

5.Acknowledgement
We are grateful to Dr. T. Mori and Dr. N.
Igarashi for their help in X-ray
diffraction experiments. We also thank toMs. S. Syoji for preparing solutions forthe experiments.
6.References[1] Kurokawa H, Fujii W, Ohmi K,Sakurai T, and Nonomura Y. (1990)
Simple and rapid purification of brevin.
Biochem Biophys Res Commun 168, 451457[2] Takemori S, Yamaguchi M, Yagi N.
Effects of adenosine diphosphate on thestructure of myosin cross-bridges: an Xray diffraction study on a single skinned
frog muscle fibre. (1995) J Muscle Res.
Cell Motil. 16, 571-577*sml@jikei.ac.jp