Guhathakurta, Piyali

Prochniewicz, Ewa

Thomas, David D.

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648a Wednesday, February 6, 20123327-Pos Board B482
Effects of Disease Causing Point Mutations on Actin’s Resilience
Ashley Brate, Ava Lin, Jonathan Crain, Ewa Prochniewicz, Jamex Ervasti,
David D. Thomas.
University of Minnesota, Minneapolis, MN, USA.
We have used spectroscopic probes to determine the structural dynamics of
actin, as affected by point mutations that are known to cause Duchenne muscu-
lar dystrophy (DMD). DMD is caused by mutations within the cytoskeletal pro-
tein dystrophin. The present study focuses on single amino acid replacements in
the N-terminal actin-binding domain (DysABD1), which result in muscular
dystrophies with a wide spectrum of severities. Though these point mutations
occur in ABD1, the affinity of dystrophin for actin is not substantially perturbed
(Henderson et al., PNAS 2010). However, affinity is only one facet of dystro-
phin’s interaction with actin. We have previously shown, using transient phos-
phorescence anisotropy (TPA), that binding of dystrophin to actin increases its
resilience, and we hypothesize that this cytoskeletal resilience is needed to act
as a mechanical buffer during contractions. Previous results have shown a cor-
relation between the TPA-measured resilience and the physiological measures
of muscle mechanical function in mice (Lin et al., J. Mol Biol 2012). In this
project, we expand this area of research by testing several known disease-
causing point mutations in DysABD1 and evaluate their effects on actin’s
resilience. We hypothesize that while affinity is not significantly affected,
mutant full length dystrophin loses its natural ability to increase the resilience
in actin as determined from TPA. Our findings will provide insight into the
pathophysiology of these disease-causing mutations - why some mutations
are more devastating than others, and why a single amino acid change can
have a large impact on dystrophin’s function.
3328-Pos Board B483
Large Scale Opening of the N Terminal Actin-Binding Domain of
Dystrophin Detected by Dipolar Electron-Electron Resonance (DEER)
Jonathan Crain, Ava Y. Lin, Brandon One Feather, Ashley Brate,
Gage Matthews, Bengt Svensson, David D. Thomas.
University of Minesota, Minneapolis, MN, USA.
We have used dipolar electron-electron resonance (DEER) to observe the struc-
tural dynamics of the calponin homology (CH) domains within the N-terminal
actin binding domain (ABD1) of dystrophin, in order to determine the confor-
mation of the ABD1 while free in solution and while actin-bound. Loss of func-
tional dystrophin causes Duchenne (DMD) and Becker (BMD) muscular
dystrophies, prompting a need for structural information about dystrophin to
aid in engineering gene therapy constructs. We have previously shown that
the ABD1 of dystrophin’s autosomal homologue utrophin opens upon actin
binding via an induced fit mechanism, suggesting a similar mechanism for
the dystrophin ABD1. Site-directed spin labeling and DEER were used to mea-
sure the distance between labels on the two CH domains, in the presence or ab-
sence of actin and at varying ionic strength. DEER spectra showed that like
utrophin, the actin-bound dystrophin ABD1 occupied a single well-ordered
conformation in which the CH domains were much more open than when the
ABD1 is free in solution. Unlike utrophin, the dystrophin ABD1 was much
more disordered when free in solution, an effect that was exacerbated by in-
creasing the ionic strength. We conclude that, similarly to utrophin, the dystro-
phin ABD1 undergoes a closed-to-open transition upon binding to actin. In
contrast to utrophin, the dystrophin ABD1 is much more disordered when
not bound to actin, suggesting that dystrophin is intrinsically less stable than
utrophin. These results provide the needed structural foundation for studying
disease-causing mutations in ABD1, and they provide a rationale for using utro-
phin in place of dystrophin in DMD therapy.
This work was funded by NIH Grants AR032961, AR057220 Core C, and
AG026160 (to D.D.T.), MDA Grant 4322 (to D.D.T.), and NIH grant
F30AG034033 (to A.Y.L.).
3329-Pos Board B484
Structural Dynamics of Actin during Active Interaction with Myosin
depends on the Isoform of the Essential Light Chain
Ewa Prochniewicz, Piyali Guhathakurta, David D. Thomas.
University of Minnesota, Minneapolis, MN, USA.
Muscle contraction results from cyclic interaction of myosin and actin, driven
by ATP hydrolysis, where the actomyosin complex undergoes transitions be-
tween weakly and strongly bound structural states. Rabbit skeletal muscle my-
osin has two isoforms of the essential light chain (ELC), designated A1 and A2.
These isoforms have significant effects on myosin’s interaction with actin, re-
sulting in higher catalytic efficiency for A1 but higher in vitro motility and
muscle shortening velocity for A2. In order to understand the structural basis
of these differences, we have used time-resolved phosphorescence anisotropy
(TPA) to investigate the effects of ELC isoforms on the microsecond dynamicsof actin during interaction with skeletal muscle myosin in the absence and pres-
ence of saturating ATP. Actin was labeled with a phosphorescent probe at
C374, and the myosin head (S1) was separated into isoenzymes containing
A1 (S1A1) and A2 (S1A2) by ion-exchange chromatography. As previously
reported, S1A1 exhibited substantially lower ATPase activity at saturating
actin but substantially higher apparent actin affinity, resulting in higher cata-
lytic efficiency. In the absence of ATP, each isoenzyme increased actin’s final
anisotropy similarly, indicating similar restriction of the amplitude of intrafila-
ment rotational motions in the strong-binding (rigor) state of actomyosin. In
contrast, in the presence of saturating ATP, the final anisotropy of actin was
substantially higher during interaction with S1A1 than with S1A2. Thus,
S1A1 was more effective in restricting actin dynamics during the active inter-
action of actin and myosin, suggesting greater population of the strong-binding
state of actomyosin. We conclude that ELC of skeletal muscle myosin modu-
lates strong-to-weak structural transitions during the actomyosin ATPase cycle
in an isoform-dependent manner, and thus modulates the contractile function of
actomyosin.
3330-Pos Board B485
A Small Chemical Mimicking Actin Binding to Myosin
Takayuki Miyanishi1, Taku Yamaguchi2.
1Nagasaki University, Nagasaki, Japan, 2Nagasaki university, Nagasaki,
Japan.
We synthesized TRA, a potent inhibitor to skeletal myosin, and its analogues,
and investigated the effect of TRA on function and structure of skeletal myosin.
ATPase assays using TRA and its analogues showed that long alkyl chain and
carboxyl group of the TRA structure was indispensable for the inhibitory effect
and the interaction with myosin structure. TRA activated the Mg2þ-ATPase ac-
tivity and decreased the amount of M.ADP.Pi formation of myosin. Myosin
head did not bind actin filament in the presence of TRA. Fluorescence measure-
ments and circular dichroism data reflected that specific structural change of
myosin heads occurred while the myosin Mg2þ-ATPase activity was activated
in the presence of TRA. All these results support the idea that TRA binds my-
osin heads and induce the actin bound state of myosin heads. TRAmay be a use-
ful chemical to mimic the actin bound state of muscle myosin. We also studied
a possible binding site of TRA on the myosin structure by using computational
modeling.
3331-Pos Board B486
High-Resolution EPR of a Bifunctional Spin Label Reveals Structural
Transitions within Myosin’s Catalytic Domain
Benjamin Binder, Ryan Mello, Rebecca Moen, David D. Thomas.
University of Minnesota, Minneapolis, MN, USA.
We present a method for obtaining high-resolution information on protein
backbone structure and dynamics using electron paramagnetic resonance
(EPR) of a bifunctional spin label (BSL) and molecular modeling. Two com-
plementary EPR techniques were employed to measure protein orientation
(conventional EPR) and intra-protein distances (dipolar electron-electron res-
onance, DEER). BSL attaches at Cys positions i and iþ4 on a helix, greatly
reducing probe mobility relative to the peptide backbone, compared to mono-
functional labels. Accurate modeling of BSL provides the coordinates re-
quired to directly relate spectroscopic data to backbone structure (both
orientation and distance), and dynamics (rotational motion). In the current
work, the motor protein Dictyostelium myosin II was used to demonstrate
this approach. We measured nucleotide-dependent structural transitions of
two key helices within the myosin catalytic domain (CD). Two double-Cys
sites were engineered, with one Cys pair located on the relay helix, and the
other on a stable helix in the upper 50kD domain. BSL on a construct with
one of these pairs was used to measure myosin orientation relative to oriented
actin. BSL on a construct with two pairs was used to measure interprobe dis-
tances. The effect of ADP binding on both orientation and distance was
clearly detected with BSL, but not with a monofunctional label. The signifi-
cance of this work is twofold: (1) A structural transition in the relay helix
upon ADP binding was clearly defined with high resolution. (2) BSL spectra
demonstrate superior resolution, compared to monofunctional spin labels,
making it possible to directly translate spectroscopic data to protein structure
and dynamics.
3332-Pos Board B487
Cardiac F-Actin has Two Functional States
Bing Sun, Maria E. Moutsoglou, John M. Robinson.
South Dakota State University, Brookings, SD, USA.
Previously, in mouse cardiac myofibrils, we showed that (i) strong cross-
bridges (XBs) prevent the exchange of cTn within the A-band and (ii) ATP-
induced release of strong XB allowed nearly uniform cTn exchange within
the thin filament. Here, we examined the mechanism for XB-dependent


Structural Dynamics of Actin during Active Interaction with Myosin depends on the Isoform of the Essential Light Chain 

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Structural Dynamics of Actin during Active Interaction with Myosin depends on the Isoform of the Essential Light Chain

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