Cooke, Roger

Franks-Skiba, Kathleen

Naber, Nariman

Pate, Ed

Purcell, Thomas J.

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Sunday, February 21, 2010 143adye-labeled lipid moieties will bring new insights into lipid organisation and
trafficking at the synapse.
Muscle: Fiber & Molecular Mechanics &
Structure I
737-Pos
Myosin Nucleotide Pocket Thermodynamics Measured by Epr Reveal
How Energy Partitioning Relates Speed to Efficiency
Thomas J. Purcell1, Nariman Naber1, Kathleen Franks-Skiba1, Ed Pate2,
Roger Cooke1.
1University of California, San Francisco, San Francisco, CA, USA,
2Washington State University, Pullman, WA, USA.
We have used spin labeled ADP to investigate the dynamics of the nucleotide-
binding pocket in myosins. In actomyosinADP the nucleotide-binding pocket
is in an equilibrium between closed and open conformations, with the open con-
formation favored in slow myosins. In rabbit slow skeletal muscle fibers, the
˛00G for the closed to open equilibrium is 3.9 kJ mol1. We found similar
values for pig ventricle myosin, chicken gizzard smooth muscle myosin, and
chicken myosin V. For faster myosins, the equilibrium shifts to favor a closed
conformation, rising to 2.7 kJ mol1 for Dictyostelium discoideum myosin
II, 1.9 kJ mol1 for pig atrial myosin, 1.1 kJ mol1 for rabbit fast skeletal
muscle fibers, andþ2.9 kJ mol1 forDrosophila fight muscle fibers.We believe
this represents a destabilization of the open actomyosinADP state in the faster
myosins, driving ADP release. van’t Hoff analysis of the temperature depen-
dence of this equilibrium reveals that the closed to open conformation has a sig-
nificant positive enthalpy and entropy,with˛00H andT˛00S of 40-50 kJmol1 for
slow myosins. Both components are reduced in this equilibrium for faster myo-
sins, decreasing to˛00H¼ 17.7 kJ mol1 and T˛00S at 25C¼ 18.8 kJ mol1 for
rabbit fast skeletal fibers, and ˛00H¼ 10.4 kJ mol1 and T˛00S at 25C¼ 7.5 kJ
mol1. Our model is that the open actomyosinADP state represents a partition-
ing point between the free energy released during themyosin catalytic cycle. Be-
cause of this partitioning, fast myosins destabilize the actomyosinADP state,
reducing the energy available to do work up until that point, but leaving more
free energy in reserve to drive ADP release. This gives a mechanism for the cor-
relation between increased speed and reduced efficiency in muscle.
738-Pos
Structural Basis for Uncoupling of Force Generation in the F506A
Dictyostelium Myosin Revealed by Time-Resolved EPR and FRET
Roman V. Agafonov1, Igor V. Negrashov1, Sarah E. Blakely1,
Margaret A. Titus1, Yuri E. Nesmelov2, David D. Thomas1.
1University of Minnesota, Minneapolis, MN, USA, 2University of North
Carolina, Charlotte, NC, USA.
We have used dipolar electron-electron resonance (DEER) and time-resolved
fluorescence resonance energy transfer (TR-FRET) to investigate the role of
the myosin relay helix in coupling between the active site and the force-gener-
ating region of myosin II. Two double-Cys Dictyostelium myosin constructs
have been engineered, and the structure of the relay helix was monitored by
measuring interprobe distances in MSL/MSL or IAEDANS/Dabcyl-labeled
myosin. Experiments were performed on WT myosin and on F506A, a func-
tional mutant that has close to normal enzymatic activity but completely lacks
motor functions (e.g. unable to move actin filaments or support cell develop-
ment). We found that the WT myosin relay helix adopts two distinct states
(straight and bent), with the bent conformation populated when ATP and
ADP.Pi analogs are bound at the active site. In contrast, binding of nucleotide
analogs had very little effect on relay helix conformation in the F506A mutant.
In addition, the width of the distance distribution was significantly larger in the
F506A compared to WT myosin, indicating loss of structural organization. Our
results demonstrate that the relay helix plays a key role in coupling of myosin
ATPase and motor activities. Loss of functionality observed in F506A myosin
can be explained by the disruption of the relay helix-relay loop interactions that
normally stabilize well-defined conformations of the myosin force-generating
region allowing it to switch between distinct structural states.
739-Pos
Subpopulation of Intermediates in Actomyosin Crossbridge-Cycle During
Sliding
Eisaku Katayama1, Yoshitaka Kimori2, Norio Baba3.
1Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 2Dept
of Ultrastr. Res, Nat’l Inst. of Neuro.& Psychi., Kodaira, Tokyo, Japan, 3Dept
of Inform. Sci., Kogakuin University, Hachioji, Tokyo, Japan.
We have been investigating conformational changes of myosin crossbridges
during in vitro sliding, utilizing quick-freeze-replication and a novel image
analysis to quantitatively compare microscopic images with the atomic modelsof various conformations. We reported that the leverarm moiety of working
crossbridges, is mostly kinked to the opposite side of ADP/Vi-bound structure
and such global configuration of myosin head resembles that whose SH1 and
2 are chemically crosslinked. Since the crystal structure of such unusual con-
figuration is not determined, we attempted to reconstruct its 3-D structure by
a special version of Single-Particle-Analysis, devised to adapt small-sized par-
ticle, utilizing very contrasty feature of metal-replicated images. With a new
procedure to manage very few view-angles, we finally obtained the 3-D enve-
lope of the myosin head with oppositely kinked leverarm, analogous to the in-
termediate structure under sliding conditions. We cut the original atomic model
of pPDM-treated-ADP-structure (1L20) into motor-domain and the leverarm,
and relocated each module at best-matching position and the orientation, to
generate a tentative model that best-fits to reconstructed envelope. We then ex-
amined whether all the images of actin-sliding crossbriges can be uniquely ex-
plained by that standard SH-crosslinked structure model. The images of actin-
attached crossbridges were classified by ‘‘2-D appearance’’ and each class-av-
erage was compared with 2-D projections of the standard structure as above. By
analyzing the orientation of motor-domain and leverarm separately, we found
that there could be several sub-populations, some matched to but some deviated
from the standard structure. It is known that two reactive thiols can be cross-
linked by a variety of bifunctional reagents of different span lengths, implying
the distance between them might be flexible. We assume that the extension of
new oppositely-kinked configuration would comprise the power-stroke and
those sub-populations might correspond to several steps during that structural
change.
740-Pos
Analysis of Conformation of the Skeletal Muscle Myosin Modified by
F2DNB Using FRET
Masafumi D. Yamada1, Koichiro Ishiyama1, Eisaku Katayama2,
Yoshitaka Kimori3, Shinsaku Maruta1.
1Dept. of Eng., Soka Univ., Hachioji, Tokyo, Japan, 2Div. of Biomol. Imag.,
Inst. of Med. Sci., The Univ. of Tokyo, Shiroganedai, Tokyo, Japan, 3Dept. of
Ultrastr. Res., Nat’l Inst. of Neurosci., Nat’l Ctr. of Neurol. and Psych.,
Kodaira, Tokyo, Japan.
Previously biochemical studies have demonstrated that the highly reactive cys-
teine residues SH1 and SH2 can be crosslinked by variety of bifunctional re-
agents with different spans (3-14 A˚) in the presence of nucleotides, suggesting
that the region is highly flexible. The SH1-SH2 region is believed to play a key
role in the conformational changes that occur in the myosin head during the
force generation coupled to ATP hydrolysis. We have previously shown that
the skeletal muscle heavy mero-myosin (HMM), which SH1-SH2 was cross-
linked by 1,5-difluoro-2, 4-dinitrobenzene (F2DNB) in the presence of ADP,
have a novel conformation using quick freeze deep etch electron microscopy
(QFDE-EM). We have also demonstrated that conformational change of the
myosin motor domain during ATP hydrolysis can be monitored by measuring
the FRET using fluorescent ATP analogue NBD-ATP. In the present study, we
analyzed the conformation of the myosin crosslinked by F2DNB using FRET
between the ATP binding site and the essential light chain (ELC) A1. We pre-
pared skeletal muscle myosin subfragment-1 (S1), which ELC was labeled by
6-bromoacetyl-2-dimethylaminonaphthalene (BD) at the Cys 177. And fluores-
cent ADP analogue NBD-ADP was trapped within the ATPase site of S1 la-
beled by BD. The FRET efficiency was estimated by measuring the change
of fluorescence intensity of BD comparing with control BD-S1. The FRET ef-
ficiency of F2DNB-S1-NBD-ADP was lower than S1-NBD-ADP state. This
suggests that the F2DNB-S1-ADP states form more kinked conformation
than S1-ADP state.
741-Pos
Switch-2 Dependent Modulation of the Myosin Power Stroke
Daniela Kathmann, Ralph P. Diensthuber, Falk K. Hartmann,
Roman Fedorov, Dietmar J. Manstein, Georgios Tsiavaliaris.
Medical School Hannover, Hannover, Germany.
Central to the mechanism of myosins are two conserved sequence motifs,
switch-1 and switch-2, which contact nucleotide and Mg2þ at the rear of the
nucleotide-binding pocket. They act as gamma-phosphate sensors, thus control-
ling hydrolysis and product release. The movement of switch-2 from a ‘closed’
to an ‘open’ conformation is translated via the relay region into a rotation of the
converter domain, which drives the force generating power stroke. To investi-
gate the molecular details of this coupling mechanism, we introduced mutation
S456Y in switch-2 of the myosin-2 motor domain from Dictyostelium discoi-
deum and analyzed the structural and functional consequences. Our kinetic re-
sults showed that the S456Ymutant lost the ability to bind effectively ATP, dis-
played a strongly decreased actin affinity in the ADP-bound state, and moved
actin filaments with highly reduced velocities. It has been proposed that the


Myosin Nucleotide Pocket Thermodynamics Measured by Epr Reveal How Energy Partitioning Relates Speed to Efficiency 

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Myosin Nucleotide Pocket Thermodynamics Measured by Epr Reveal How Energy Partitioning Relates Speed to Efficiency

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