Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

15A/2003G143

CONFORMATIONAL CHANGES OF PROCESSIVE MYOSIN MOTORS(MV, MVI) BY SMALL-ANGLE X-RAY SCATTERINGYasunobu SUGIMOTO1, Osamu SATO2, Shinya WATANABE2, Mitsuo IKEBE2,Yasunori TAKEZAWA1 and Katsuzo WAKABAYASHI*1
1Division of Biophysical Engineering, School of Engineering Science, Osaka University,Toyonaka, Osaka 560-8531, Japan2
Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655,
USA
Introduction
The myosin motors can produce a force and movement
on actin filaments using the chemical energy ofhydrolysis of ATP.
Myosin V is one of theunconventional myosins like as Myosin VI. Myosin V
moves toward the barbed (+) end of actin filaments, but
myosin VI moves toward the pointed (-) end. In order toclarify the mechanism of the opposite directional motion
on the actin filaments, we have investigated the structuralchanges of the subfragmet-1 (S1) of myosin V and VI
which are related with an ATP hydrolysis using X-ray
solution scattering techniques.
Experimental
Recombinant myosin V S1 with two IQ motifs (MVS1IQ2) and myosin VI S1 (MVI-S1) were obtained from
Sf9 cells by baculovirus expression system. MV-S1IQ2and MVI-S1 constructs were purified and collected
through a column chromatography. Purified skeletal
muscle myosin subfragment-1 (MII-S1) was also used asa reference. The X-ray solution scattering experiments
were done at 20°C at the BL15A1 using the small-angle
diffractometer. All X-ray scattering data were collected
as a function of scattering vector length (S=2sinθ/λ) witha 1D-PSD. The protein concentration (c) was varied inthe range of 2 to 7mg/ml.

Results and Discussion
The Guinier plots of the scattering data from the MVS1IQ2, MVI-S1 and MII-S1 samples with or without
nucleotide gave all straight line, indicating no aggregate
in any solution. The radius of gyration (Rg) value and thezero-angle scattering (I(0)) were calculated from theGuineir plots on each samples. The Rg and I(0)/c versus
c plots were linear and their values extrapolated to zeroprotein concentration were determined. All samples had
almost the identical molecular weight from their I(0)/cvalues, constitent with SDS-PAGE data. Fig. 1 shows theconcentration dependence of Rg from various samples.
The Rg value of MV-S1IQ2 was 48.6Å and the Rg ofMV-S1IQ2 in the MgATP solution decreased increasedby ~2Å (Fig.1A). These values were very similar to thatof MII-S1 with and without nocleotide. On the otherhand, the Rg of MVI-S1 increased about 3Å in theMgATP solution (Fig.1B). The most probable cause of
the change in Rg would come from motion of lever-arm
portion. These results showed that the directionality ofmyosin motors on actin filaments closely relates to thedirectional structural changes of their lever arm: MV-S1
becomes to more compact and MVI-S1 becomes moreelongated in the MgATP solution. The changes of Rg ofMV- and MVI-S1 with ADP were small. Experiments ofseveral nucleotide analogues bound to MV-S1 and MVIS1 suggested that myosin V and myosin VI changed theirshape in M.ATP state before M.ADP.Pi state in theATPase cycle.

Figure 1 Concentration dependence of Rg calculated
from Guinier plots of X-ray scattering intensities ofmyosin V and myosin VI construct solution.

* waka@bpe.es.osaka-u.ac.jp

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