Mutations in the ?-myosin rod cause myosin storage
myopathy via multiple mechanisms
Thomas Z. Armel and Leslie A. Leinwand1
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309
Edited by Jonathan G. Seidman, Harvard Medical School, Boston, MA, and approved February 27, 2009 (received for review January 6, 2009)
Myosin storage myopathy (MSM) is a congenital myopathy charac-
majority of type I muscle fibers, and has been linked to 4 mutations
in the slow/cardiac muscle myosin, ?-MyHC (MYH7). Although the
majority of the >230 disease causing mutations in MYH7 are located
in the globular head region of the molecule, those responsible for
MSM are part of a subset of MYH7 mutations that are located in the
?-helical coiled-coil tail. Mutations in the myosin head are thought to
affect the ATPase and actin-binding properties of the molecule. To
date, however, there are no reports of the molecular mechanism of
pathogenesis for mutations in the rod region of muscle myosins.
Here, we present analysis of 4 mutations responsible for MSM:
L1793P, R1845W, E1886K, and H1901L. We show that each MSM
mutation has a different molecular phenotype, suggesting that there
are multiple mechanisms by which MSM can be caused. These mech-
anisms range from thermodynamic and functional irregularities of
individual proteins (L1793P), to varying defects in the assembly and
H1901L). In addition to furthering our understanding of MSM, these
observations provide the first insight into how mutations affect the
rod region of muscle myosins, and provide a framework for future
studies of disease-causing mutations in this region of the molecule.
a dimer of myosin heavy chains (MyHC) with 2 globular heads
attached to a long, ?-helical coiled-coil region known as the
myosin rod. Although the head region is responsible for myosin’s
ATPase and actin binding properties, the rod region functions to
incorporate myosin into bipolar thick filaments through charge
based interactions between adjacent rods (1–3). ?-myosin, which
is expressed in the adult heart and slow skeletal muscle fibers, is
of particular clinical importance because to date ?230 disease
causing mutations have been found in the molecule (4). The
majority of mutations (65%) are located in the head region and
result in cardiomyopathies, many of which have been well
characterized (5). However, several mutations have also recently
been described in the rod region of myosin that result in a variety
of diseases (6, 7). Because of the disparate roles that the head
and rod regions of myosin play, these mutations are thought to
in the rod region of muscle myosins have been characterized.
We sought to investigate a subset of mutations located in the
myosin rod that are responsible for the disease myosin storage
myopathy (MSM). Also known as hyaline body myopathy, MSM
is a rare, congenital myopathy with variable inheritance that is
characterized by the presence of subsarcolemmal accumulations
of myosin in the majority of type-I skeletal muscle fibers (8, 9).
Clinically, patients exhibit variable age of onset ranging from
also vary, but typically include slowly progressive muscle hyper-
tonia, scapularperoneal weakness, and respiratory insufficiency
(6). MSM has been associated with 4 missense mutations in the
MYH7 gene, which encodes slow/?-cardiac myosin heavy chain
(MyHC). All 4 mutations responsible for MSM, L1793P,
R1845W, E1886K, and H1901L, are located in the distal rod
region of the protein and have been postulated to result in
yosin is the molecular motor of muscle and is the major
component of the thick filament. In vivo, myosin exists as
pathogenesis by perturbing the lateral interactions between
coiled-coils (Fig. 1A) (8–10).
The ?-helices that compose the coiled-coil myosin rod are
characterized by a repeating heptad of residues (denoted a–g),
that in turn is part of a larger 28 aa repeat composed of 14
positively charged residues followed by 14 negatively charged
residues (Fig. 1B) (1–3). It is the interaction of these charged
repeats, in proper register, which is responsible for formation of
the thick filament. Also crucial to the association of the rods is
a region of 29 aa known as the assembly competent domain
(ACD), which our laboratory has demonstrated to be necessary
for the formation of myosin rod assemblies in vitro (11) .
In an ?-helical coiled-coil, residues in the a and d position are
typically hydrophobic and create a seam along the interface of
the coiled-coil, whereas residues in the e and g positions interact
electrostatically to stabilize the coiled-coil. Residues in the outer
positions such as b, c, and f are typically charged and mediate the
interactions between coiled-coils (Fig. 1B) (12–14). Of the
mutations responsible for MSM, 3 (R1845W, E1886K, and
H1901L) are located in the f position, whereas 1 (L1793P) is
located in the d position. Interestingly, L1793P, R1845W, and
H1901L are associated with MSM in the absence of any overt
cardiomyopathy, even though the protein is abundant in both
heart and skeletal muscle. E1886K is the only mutation that is
responsible for MSM that also results in hypertrophic cardio-
myopathy, and is located in the ACD region of the rod. Given
that these mutations have been predicted to alter the ability of
myosin to properly assemble into stable thick filaments, we
sought to experimentally characterize their effects by perform-
ing a wide variety of structural and functional assays. Here, we
show that MSM mutations expressed in the C-terminal fragment
of the rod, light meromyosin (LMM), cause structural, thermo-
dynamic, and functional differences that correlate with varying
phenotypes. This provides the first description of how disease
causing mutations affect the rod region of a muscle myosin, and
sheds lights on the pathogenesis associated with these mutations.
The entire LMM region of ?-MyHC (amino acids 1231–1938)
was cloned and tagged with T7 and 6X-His at the N terminus for
recombinant expression. WT and mutant proteins were gener-
ated and purified as described in Materials and Methods, and
had an expected electrophoretic mobility of ?82 kDa. SDS/
PAGE analysis confirmed that all proteins were purified to
MSM Mutations Alter Protein Stability Without Affecting Secondary
Structure. To determine whether the ability of LMM to self-
assemble could be affected by structural defects within the
data; and T.Z.A. and L.A.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
April 14, 2009 ?
vol. 106 ?
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molecule, circular dichroism (CD) spectroscopy was used to
ascertain whether mutations altered the ?-helical coiled-coil
secondary structure of LMM. As an ?-helical coiled-coil, the
LMM region of the myosin rod displays a distinct CD spectra,
with characteristic minima at 222 nm and 208 nm (15). The
spectrum for wild-type LMM displayed the expected pattern, as
did all 4 of the mutant proteins studied (S1). All of the mutant
proteins also displayed similar ?-helical content to WT, each
being ?88%. The ?222/208ratios, which report on the ability of the
?-helices to form coiled-coils, were all ?1.11, indicating that
coiled-coil formation is not disrupted (Table 1) (13, 14, 16).
folded, we next determined the effect of these mutations on the
thermal stability of LMM by monitoring [?]222as the tempera-
ture was increased from 5 °C to 90 °C. WT and all 4 mutant
proteins were modeled as a 2-state transition of monomer from
between the folded and unfolded forms (17). The calculated
thermal midpoints of unfolding for R1845W, E1886K, and
H1901L are slightly decreased from WT, whereas L1793P
displays a relatively larger decrease in thermal stability (Fig. 2).
Additionally, although the biophysical parameters for R1845W
and E1886K are nearly indistinguishable from WT, L1793P has
a ??G at 37 °C of ?0.62 kcal/mol and displays decreases of
?18% for ?H, ?S and ?Cp. H1901L displays an intermediate
phenotype, with a ??G at 37 °C of ?0.26 kcal/mol and an ?8%
decrease in ?H, ?S, and ?Cp (Table 1). Taken together, these
data indicate that the secondary structure of LMM is not altered
by any of the mutations studied, whereas 2 of the mutations
causing MSM (L1793P and H1901L) do alter the thermody-
namic stability of the molecule.
MSM Mutations Affect Filament Formation. We next sought to
examine whether these disease causing mutations do indeed
affect the ability of LMM to self-assemble. To monitor this
process, LMM was diluted from 300 mM NaCl where it exists
stably as a coiled-coil, to 150 mM NaCl where it will self-
by 90° light scattering. 90° light scattering is an assay that follows
the assembly of LMM in real time based upon the size of the
particles scattering light, and represents a tractable model for
studying a variety of mutations in the myosin rod (18, 19). By this
assay, WT LMM quickly assembled, with the majority of the
reaction completed after 8–10 min. R1845W, E1886K, and
H1901L appear to assemble at rates comparable to WT, but the
extent to which the reactions occur is truncated to ?60% of WT
in each case. L1793P assembles much more slowly than WT, with
a lag phase before assembly that effectively doubles the amount
of time required for the majority of the protein to self-associate.
In addition, the extent to which the reaction occurs reaches
?50% of that for WT (Fig. 3).
To further investigate the ability of LMM to self-assemble, we
tested the ability of WT and each of the mutant proteins to form
paracrystals. Paracrystals are well-ordered protein assemblies
that provide an established model for thick filament formation
(1, 20–26). Similarly to light scattering experiments, proteins
were dialyzed from a high salt buffer into a buffer with physi-
ological salt concentration allowing them to assemble into
well-ordered arrays. Paracrystals were then analyzed by electron
microscopy (EM). WT LMM formed well-ordered paracrystals
with a periodicity of 14.0 nm (?0.44 nm for n ? 20 paracrystals;
S2). This value is consistent with reported values for LMM
paracrystal periodicity and corresponds well to the 14.3 nm axial
spacing of full-length ?-MyHC found in bipolar thick filaments
(1, 20, 21, 24, 26). Paracrystals formed from L1793P, R1845W,
E1886K, and H1901L LMM all had similar periodicities to WT,
indicating that these mutations do not alter the gross morphol-
ogy of the assemblies (S3).
MSM Mutations Alter Filament Properties. Although the periodicity
of paracrystals formed from WT and mutant proteins is similar,
we wanted to further examine these assemblies to assess whether
their stability is affected by the mutations being studied.
Paracrystals were formed as before, and limited proteolysis was
carried out by digestion with a low concentration of porcine
trypsin. Reactions were quenched at various time points out to
90 min using soybean trypsin inhibitor, and the reactions were
analyzed via SDS/PAGE to visualize degradation (Fig. 4A). The
band intensity of the full length LMM was then measured and
model (Fig. 4B). WT protein is shown to be relatively stable over
time, with ?80% of the full length protein still intact after 90
min. L1793P LMM shows a similar pattern to WT, although it
is degraded slightly more rapidly. E1886K LMM, however, is
much more readily proteolyzed, with the majority of the full
length protein digested by the 10-min time point. Interestingly,
(A) Schematic of ?-MyHC structure. The globular head region and N-terminal
portion of the myosin rod which comprise heavy meromyosin are shown in
dark gray. The C-terminal LMM region of the rod is shown in light gray with
position of amino acids within the heptad is denoted by a–g. Three MSM
mutations (R1845W, E1886K, and H1901L) are located in the outer f position,
whereas 1 (L1793P) is located in the inner d position.
Location of MSM mutations in the coiled-coil rod region of ?-MyHC.
Table 1. Biophysical data for myosin tail constructs
www.pnas.org?cgi?doi?10.1073?pnas.0900107106Armel and Leinwand
R1845W and H1901L LMM appear to be more stable than WT.
Careful analysis of the gels, however, shows degradation of both
R1845W and H1901L at later time points (30% of R1845W and
18% of H1901L are degraded at 90 min compared with 26% of
WT). Because degradation begins later for paracrystals formed
from these 2 proteins, when these data are fit to an exponential
suggest that the kinetics of R1845W and H1901L LMM prote-
olysis occur more slowly than WT and that paracrystals formed
from these mutant proteins may be slightly more stable, whereas
L1793P and E1886K LMM are more readily degraded.
We next carried out molecular sizing experiments, using
dynamic light scattering (DLS) to ascertain whether qualitative
differences exist between paracrystals formed from WT and
mutant LMM. DLS is a technique that measures the size
(hydrodynamic radius) of a particle by determining the fre-
quency shift of coherent light directed at that particle. Our DLS
experiments show that control protein that was not assembled
into paracrystals had a measured hydrodynamic radius of 22 nm,
whereas paracrystals formed from WT LMM had hydrodynamic
radii of ?29,500 nm. L1793P and E1886K LMM formed
paracrystals that were uniform in size distribution, with a single
species present that was similar in size to WT. Paracrystals
formed from R1845W and H1901L LMM also had a uniform
distribution, but both formed assemblies that were much larger
(Fig. 5). It should be noted, however, that because this size is at
the limit of detection for the device used, the actual size is likely
to be larger than the measured size for each mutant.
To date, all reported instances of MSM are associated with 1 of
4 mutations in the LMM region of the ?-MyHC rod (8–10,
27–32). By using an array of assays to biochemically and bio-
physically characterize the effects that these mutations have on
molecular phenotype. Because all of these mutations are ulti-
mately responsible for the same disease phenotype, our results
suggests that there are several possible ways in which mutations
in the LMM region of ?-MyHC can adversely affect the ability
of the protein to form stable, functional bipolar thick filaments.
The assembly of muscle filaments is a multistep process that
involves both the proper folding of ?-helices into coiled-coils,
and the assembly of these coiled-coils, in proper register, into
filaments. Our findings indicate that defects in either one of
these steps can result in improper filament formation leading to
L1793P. The L1793P mutation results in the largest thermody-
namic differences from WT protein of any of the mutations
studied. L1793P LMM has a Tmthat is 1.5 °C lower than wild
type, and the lower ?G at 37 °C suggests that under in vivo
conditions the mutant protein is appreciably less stable. Surpris-
ingly, our findings show no difference in secondary structure.
followed as a function of temperature by measuring ?222, which monitors ?-helical structure, as the temperature was gradually increased. All experiments were
performed in the same high salt buffer as for far-UV CD to prevent protein assembly, and were done at 4 °C. Observed ?222data (black) were fit to a theoretical
melting curve (light gray), and are plotted on the left axis. The residuals (dark gray), calculated as the difference between the observed and the fit data at each
point, are plotted on the right axis and are ?0 for each melt.
Thermal denaturation of the myosin rod reveals differences in protein thermodynamics. The transition of LMM from a folded to an unfolded state was
EDTA, 1 mM TCEP) was observed for 120 s before the addition of protein to obtain a baseline for scattering. An equal volume of either WT (black) or mutant
(gray) LMM in high salt buffer was then injected into the sample chamber, diluting the sample to a final concentration of 150 mM NaCl and 100 nM protein to
initialize self-assembly. Reactions were followed for 40 min before the addition of 5M salt to return the sample to a final salt concentration of 300 mM to
demonstrate the reversibility of the reaction. The intensity of the 90° light scattering is plotted in arbitrary units versus time.
Real-time self-assembly of LMM shows MSM mutants are assembly defective. The amount of 90° light scattering for no salt buffer (10 mM TES, 3.5 mM
Armel and LeinwandPNAS ?
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Proline is known to be detrimental to the secondary structure of
?-helices, and is not found at the d position in the rod of any
muscle myosin isoform from any species (4, 33, 34). Although
some computational algorithms such as COILS and PAIRCOIL
predict this mutation to have a negative effect on secondary
structure, our CD results reveal no difference in the ?-helical
content of mutant protein when compared with WT. Although
there is no detectable structural difference, the instability of
L1793P LMM does appear to have a functional impact. By using
90° light scattering to monitor the reaction, we show that the
L1793P mutation alters the ability of LMM to assemble. The lag
phase present in the initial stages of L1793P LMM self-assembly
suggests that the nucleation of new filaments is less favored,
presumably because of the increased instability of the molecule.
This interpretation is also consistent with our proteolytic studies,
which show that the paracrystals that form, although similar in
Taken together, these data suggest that replacing the L1793
amino acid with a proline destabilizes the dimer interface under
conditions similar to those found in vivo, and that this instability
affects the ability of LMM to properly assemble.
R1845W. Located in the outer f position of the ?-helix heptad, the
R1845W mutation is expected to disrupt interactions between
coiled-coils without disturbing the structure of the molecule.
Predictably, R1845W LMM is nearly indistinguishable from WT
in both secondary structural characteristics and biophysical
parameters. Functional assays, however, reveal that mutating the
normally charged R1845 residue to a bulky, hydrophobic tryp-
tophan results in an interesting phenotype. Our 90° light scat-
tering assay shows that, although it assembles at a similar rate to
WT, R1845W LMM does not appear to be able to assemble to
the same extent. Because the signal is proportional to the size of
the particles responsible for the light scattering, one would
expect structures formed from this mutant protein to be smaller
in size than those formed from WT. Results, however, from
molecular sizing experiments performed by dynamic light scat-
tering show that the structures formed by R1845W LMM are
actually much larger that those formed by WT. These data are
substantiated by our limited proteolysis experiments, which
demonstrate that paracrystals of R1845W LMM are more stable
than those of WT, although they appear morphologically similar
by EM. This suggests that for R1845W LMM we are observing
the formation of fewer assemblies that are much larger in size
than those normally formed. These results imply a model
whereby the R1845W mutation alters the interactions between
filaments such that their assembly is less constrained, causing the
formation of abnormally large, degradation resistant structures.
E1886K. Of the 3 MSM mutations in the f position of the heptad
repeat, E1886K is expected to have the most severe phenotype
because it is located in a portion of the rod, the ACD, which is
known to be critical for the proper assembly of myosin filaments
(11). Similarly to R1845W, E1886K LMM displays no discern-
able differences in secondary structure or biophysical parame-
ters from WT, as would be expected. The mutation does,
however, have a strong functional impact on filament formation.
Results from 90° light scattering show that, like R1845W,
E1886K LMM has a decreased ability to assemble to the same
extent as WT LMM, although the initial rates of assembly are
similar. Unlike R1845W LMM, although, E1886K LMM forms
structures that are measured by dynamic light scattering to have
a hydrodynamic radius similar to WT. Interestingly, even though
EM of E1886K LMM paracrystals does not reveal any overt
morphological phenotype, our limited proteolysis studies show
that paracrystals formed from this mutant protein are much
more readily degraded than those formed from WT protein.
Proteolytic cleavage by trypsin is known to occur at lysine and
arginine residues, so it is possible that the increased proteolysis
is due to inserting an exogenous cleavage site. However, control
experiments performed by inserting a single lysine residue into
represent different time points for digestion (lane 1, 0 min; lane 2, 1 min; lane 3, 3 min; lane 4, 5 min; lane 5, 8 min; lane 6, 10 min; lane 7, 30 min; lane 8, 45 min;
data to an exponential decay curve. Data are plotted as relative intensity of the full length LMM band versus time. The band intensity for each protein at the
zero time point is defined as 1, and all other band intensities for the protein are plotted with respect to that value.
MSM mutants affect the proteolytic stability of LMM paracrystals. (A) Time course of limited tryptic proteolysis of LMM. Lanes numbered 1 through 11
Paracrystals were formed by diluting the protein from high salt buffer to low
salt buffer, similarly to static light scattering experiments, but to a final
which was measured before assembly as a control.
R1845W and H1901L LMM form drastically larger paracrystals.
www.pnas.org?cgi?doi?10.1073?pnas.0900107106Armel and Leinwand
another site in LMM do not result in a significant difference
from WT (data not shown). Additionally, 125 lysine and arginine
residues exist in the WT LMM being studied, 31 of which are
located in the f position of the coiled-coil. These potential
cleavage sites do not appear to be targets of excessive proteolytic
cleavage, suggesting that the location of the cleavage site is
important. Taken together, our data intimate that mutating the
negatively charged E1886 residue to a positively charged lysine
disrupts an important charge based interaction in a critical
region of the molecule. This appears to cause the protein to form
filaments that are packed together less tightly, allowing for
proteolytic cleavage by trypsin in our assay. The altered packing
of the filaments may also destabilize them, explaining why fewer
filament are observed in our 90° light scattering assay.
H1901L. The third MSM mutation located in the f position,
H1901L replaces a positively charged amino acid, presumably
involved in electrostatic interactions between adjacent coiled-
coils, with a small, neutrally charged amino acid. Although we
did not expect any structural or biophysical differences owing to
this mutation, the Tmof the protein did decrease by 0.7 °C and
this was associated with a ??Gat 37 °C of ?0.26 kcal/mol,
indicating that the mutation does decrease the stability of the
molecule. Functionally, the H1901L mutation appears to affect
the protein in much the same manner as R1845W. 90° light
scattering assays with H1901L LMM show that the initial rate of
assembly is unaffected, whereas the extent of assembly is dimin-
ished compared with WT. Molecular sizing experiments are also
similar, showing that paracrystals formed from H1901L LMM
are much larger than those formed from WT, whereas the
morphology as visualized by EM is unaffected. Additionally, the
H1901L mutation behaves similarly to R1845W in our tryptic
digestion assay, with paracrystals formed from H1901L LMM
appearing to be more stable than those formed from WT
protein. Our interpretation of these data, then, is much the same
as for R1845W. Although the H1901L mutation does slightly
decrease the stability of the molecule, this does not appear to
drastically affect its ability to form filaments. In fact, mutating
H1901 to a lysine appears to alter the interactions between
filaments such that larger, more stable structures are formed.
The picture that emerges from our work is that there are
several possible pathways by which mutations in the rod portion
of a muscle myosin can result in the same disease. MSM can be
caused by thermodynamic instabilities that drastically decrease
the ability of the protein to properly assemble, such as with
L1793P, or by charge changes that interrupt the interaction
between adjacent proteins and destabilize the filaments, such as
with E1886K. In vivo, these proteins may form thick filaments
that are less stable or more easily damaged, leading to sarco-
meric disarray. MSM can also be caused by the loss of important
charged amino acids, as is the case with R1845W and H1901L,
which result in the formation of atypically large, unusually stable
filaments. In vivo, these proteins may form thick filaments that
are not always properly incorporated into the sarcomere or are
not properly degraded, resulting in aberrant accumulations of
myosin in the muscle fibers. It should be noted that although our
data support these hypotheses, the possibility cannot be elimi-
nated that the observed differences are epiphenomena. Our
model will only be completely substantiated when these muta-
tions, along with other mutations having similar biophysical
profiles, are investigated in vivo. Further work will also be
needed to investigate the cause of the tissue specificity of the
MSM disease phenotype, but future studies into the pathogen-
esis of mutations in the myosin rod should benefit from our
analysis. Finally, it will be important to determine why the
mutations studied here result specifically in MSM whereas other
mutations result in a host of other disease phenotypes, and what
functional impact these mutations have on the muscle in vivo.
Materials and Methods
LMM Expression Constructs. Wild type MYH7 LMM (amino acid residues
1231–1938) was cloned into the pUC18 expression vector and N-terminal T7
and 6X-His tags were added. Site-directed mutagenesis was then performed
using inverse PCR to generate the L1793P, R1845W, E1886K, and H1901L
mutations. Positive clones were verified by sequencing and were then sub-
cloned into the pET3a expression vector (New England Biolabs), using XhoI
and SpeI. The cloning process resulted in 3 additional amino acids (TSC) being
added to the C terminus of the protein
Purification of Wild-Type and Mutant LMM Proteins. Wild-type and mutant
plasmids were transformed into BL-21 cells and expression was induced with
1 mM IPTG. Cells were harvested and protein was extracted using B-PER
pyrophosphate, 100 mM NaCl, 3.5 mM EDTA at pH 8.5. Dialyzed protein was
then additionally purified by anion exchange chromatography on an AKTA
Purifier with 1 mL of HiTrap Q HP columns (GE Lifesciences). Fractions con-
taining LMM were analyzed via SDS/PAGE gel for purity and pooled. Protein
was then concentrated with Amicon Ultracel 50k centrifuge columns (Milli-
Circular Dichroism Analysis. Wild-type and mutant LMM proteins were dia-
lyzed into 10 mM TES, 300 mM NaCl, and 3.5 mM EDTA overnight at 4 °C,
diluted to ?0.3 mg/mL, reduced with 1 mM TCEP and degassed. Circular
dichroism (CD) was measured using a Jasco J-810 spectropolarimeter (Jasco)
with constant N2flushing. A Peltier temperature control device was used to
used. Spectra were determined from 250 nm to 190 nm at a sensitivity of 100
mdeg, with a 0.2-nm data pitch, a continuous scanning speed of 50 nm/min,
and averaged ?6 accumulations for each protein. Buffer spectra were col-
concentrations were determined via uBCA assay, and the mean residue molar
ellipticity was calculated using the equation,
??? ? ?obsmrw/10/c
where ?obsis the observed ellipticity in millidegrees, mrw is the mean residue
peptide concentration (in milligrams per milliliter) (35). The percentage ?-he-
lix for each protein was determined using the equation,
%?-helix ? ((??208? 4,000)/29,000) * 100
where ?208is the mean residue molar ellipticity at 208 nm.
of wild-type and mutant LMM was monitored at 222 nm to follow ?-helical
secondary structure during temperature-induced denaturation. Data were
collected at 0.5 °C intervals at a scan rate of 60 °C per hour from 4 °C to 90 °C.
The change in mean residue ellipticity, ?, as a function of temperature was
modeled using a nonlinear least squares algorithm assuming the 2-state
transition of a monomer from a folded to an unfolded state with a change in
heat capacity, ?Cp, between the folded and unfolded forms (17). A detailed
each data point by taking the difference between the fit mean residue
ellipticity and the experimentally determined mean residue ellipticity at that
Static Light Scattering. The ability of wild-type and mutant protein to self-
assemble was measured in real-time by monitoring 90° light scattering, using
a PTI QM-2000–6SE fluorescence spectrometer (Photon Technology Interna-
tional) at 25 °C. Excitation was at 320 nm with a 2-nm slit width, and emission
was measured at 320 nm with an 8-nm slit width. Measurements were taken
for buffer (10 mM TES, 3.5 mM EDTA, 1 mM TCEP, pH 7.3) for 2 minutes to
obtain a baseline for scattering, at which point an equal volume of 200 nM
protein in high salt buffer (10 mM TES, 300 mM NaCl, 3.5 mM EDTA, 1 mM
TCEP, pH 7.3) was added, diluting the protein to 150 mM NaCl and allowing
for self-assembly at a final protein concentration of 100 nM. Assembly reac-
tions were followed out to 40 min before 5M salt was added to return the
concentration to 300 mM. Data were analyzed using Kaleidagraph (Synergy
Software) with background buffer scattering subtracted from each reading.
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Paracrystal Formation and Visualization. Paracrystals were formed for wild- Download full-text
Paracrystals were adsorbed onto glow-discharged, carbon-coated, 400 mesh
grids for 60 s, negatively stained with 2% uranyl acetate, and rinsed with
dialysis buffer. Electron micrographs were imaged using a Phillips CM10 TEM
at 80 kV. Image analysis was performed using GNU Image Manipulation
Program software (Free Software Foundation).
Limited Trypsinization. LMM was assembled as described for paracrystal for-
mation, reduced with 1 mM TCEP, and protein concentrations were deter-
as described in ref. 36. Briefly, paracrystals were treated with porcine trypsin
at 0.002 mg/mL and incubated at 30 °C. The reaction was quenched at various
times by mixing with 40? excess soybean trypsin inhibitor. Samples were run
on 4–20% SDS/PAGE gels, stained with Imperial Protein Stain (Thermo Scien-
tific) and imaged with a Li-COR Odyssey infrared imager (Li-COR).
described for static light scattering. Proteins were diluted to a final concen-
tration of 200 nM and incubated at room temperature for 45 min to allow for
complete assembly. Molecular sizing experiments were performed with a
DynaPro (Wyatt Technology) set to 10% laser power at a wavelength of 824.2
nm. All experiments were done at room temperature, using a 5-s acquisition
time. Data analysis was performed using the Dynamics software package
(Wyatt Technology) and hydrodynamic radii were estimated using the soft-
ware’s Coils model.
Supporting Information. Detailed methods, along with CD and paracrystal
data, can be found in SI.
the L.A.L. laboratory for invaluable support, discussion, and suggestions; S.
Kwok and B. Hirsch at the University of Colorado Health Sciences Center
Biophysics core for technical advice and support; M. Stowell for editorial
assistance and advice; and M. Buvoli for assisting with the preparation of the
manuscript. This work was supported by National Institutes of Health Grant
5R01 HL085573-01 (to L.A.L.) and a National Inistitutes of Health Molecular
Biophysics Training Grant T32GM065103 (to T.Z.A.).
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www.pnas.org?cgi?doi?10.1073?pnas.0900107106Armel and Leinwand