From the Laboratory of Physical Biology, NIAMS,
National Institutes of Health, Bethesda, Maryland 20892 and
§ Department of Chemistry and Biochemistry, University of
Texas, Austin, Texas 78712
Received for publication, September 28, 2000, and in revised form, November 13, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The extension of the PEVK segment of the giant
elastic protein titin is a key event in the elastic response of
striated muscle to passive stretch. PEVK behaves mechanically as an
entropic spring and is thought to be a random coil. cDNA sequencing
of human fetal skeletal PEVK reveals a modular motif with tandem
repeats of modules averaging 28 residues and with superrepeats of seven
modules. Conformational studies of bacterially expressed 53-kDa
fragment (TP1) by circular dichroism suggest that this soluble protein contains substantial polyproline II (PPII) type left-handed helices. Urea and thermal titrations cause gradual and reversible decrease in
PPII content. The absence of sharp melting in urea and thermal titrations suggests that there is no long range cooperativity among the
PPII helices. Studies with solid phase and surface plasmon resonance
assays indicate that TP1 interacts with actin and some but not all
cloned nebulin fragments with high affinity. Interestingly, Ca2+/calmodulin and Ca2+/S100
abolish nebulin/PEVK interaction. We suggest that in aqueous solution,
PEVK is an open and flexible chain of relatively stable structural
folds of the polyproline II type. PEVK region of titin may be involved
in interfilament association with thin filaments in a
calcium/calmodulin-sensitive manner. This adhesion may modulate titin
extensibility and elasticity.
The monumental sequencing work of Labeit and Kolmerer (1) has
revealed the complete domain organization of the giant elastic protein
titin. The bulk of cardiac titin consists of predominately two types of
sequence motifs: immunoglobulin (Ig) and fibronectin arranged in
three levels of motifs (repeats, superrepeats, and segments). In
addition to these well characterized domains, a novel motif consisting
of mainly four amino acid residues, Pro, Glu, Val, and Lys, is
discovered in the elastic I band region of titin. The length of this
PEVK segment varies among muscles, ranging from 183 residues in the
human cardiac titin to 2174 residues in human soleus skeletal muscle.
Differential splicing of the titin transcripts in the PEVK region as
well as in an adjacent tandem Ig segment near the A band produce these
size isoforms of titin (1, 2). Since the selective expression of titin size isoforms imparts distinct elasticity to skeletal and cardiac muscles, with the muscle expressing longer titin being more compliant (3), the observed length variation of PEVK and tandem Igs in titin
isoforms immediately suggests the possibility that either or both
segments may be the elastic elements. Labeit and Kolmerer (1)
speculated that the PEVK region, with a predicted nonfolded polypeptide, is the key elastic element of titin. The concept of
reversible unfolding and folding of Ig domains was considered unlikely,
on the ground of the thermodynamic stability of Ig domains (4, 5).
Recent works on the elasticity of single titin molecules (6), single
myofibrils (7), and single fibers (8, 9) revealed that,
stretched modestly, sarcomere elasticity can be explained by the
straightening of tandem Ig segment (without unfolding), followed by the
extension of a permanently unfolded PEVK segment (10).
To further evaluate molecular theories of titin elasticity, studies of
the conformation and stability of different classes of titin domains
are essential. The systematic NMR studies of expressed single Ig and
fibronectin domains by Pastore and coworkers (11) have provided
insightful structural details and a possible model of force generation.
In contrast, no molecular characteristics of the PEVK region have yet
been described. We report the initial sequence and molecular analysis
of a proline-rich region that we identified independently by screening
expression libraries of human fetal skeletal muscle with anti-titin
antibodies. One 2.5-kb1 clone
was found to consist of tandem repeats of a fundamental module that
averages 28 residues, mostly of PEVK residues. This sequence is
classified as a PEVK segment by its high homology with human soleus
PEVK. Conformational studies of a PEVK fragment (designated as TP1,
containing 16 PEVK modules, 468 amino acid residues) indicate that PEVK
is an open and flexible chain with stable structural folds, perhaps of
left-handed polyproline II helices, but contains little, if any,
Cloning and Sequencing of Human Fetal Skeletal Muscle
PEVK--
A Expression and Purification of PEVK Fragment PT1--
A 1.4-kb
open reading frame derived from hfT11 was subcloned into a pET3d
plasmid by digesting Bluescript clone with HinfI and ligated
to pET 3d, essentially as described in (12). The expressed protein, TP1
(51,467,469 residues), was purified from a 4-liter culture of
BL21(DE3)pLysS host cells transformed with pET3d HinfI
plasmid that was incubated at 37 °C for 3.5 h upon isopropyl-1-thio- Protein Analysis--
Polyacrylamide gel electrophoresis was
done in 12% Laemmli gel. Western blot was done with electrophoretic
transfer on nitrocellulose membrane (S&S BA85) in a Bio-Rad semidry
electroblotter at 1.5 mA/cm2 for 60 min using a buffer
system modified from Khyse-Andersen (13) Briefly, the blotting assembly
was set on the anode electrode plate of a semidry transfer unit
(Bio-Rad) in the following order: two sheets of filter paper (GB002;
Schleicher & Schüll) presoaked in anode buffer 1 (300 mM Tris-Cl, 0.05% SDS, 10% methanol, 10 mM
Blots were stained with anti-titin monoclonal antibodies, followed by
horseradish peroxidase-conjugated secondary antibodies (rabbit
anti-mouse IgG, IgM, and IgA (H + L)). Monoclonal antibodies against
rabbit skeletal titin were prepared previously in this laboratory by
standard procedures. Monoclonal antibody RT11, an IgM, was found to
react with the PEVK segment and was used to monitor the expression and
purification of TP1 by Western blots.
The concentration of TP1 was determined spectroscopically with a
calculated extinction coefficient at 280 nm of 0.025 for 1 mg/ml.
Stoke's Radius Determination of TP1--
Stoke's radius of TP1
was estimated by the method of Laurent and Killander (14). A 10 × 300-mm Superose 6 HR (Amersham Pharmacia Biotech) equilibrated with
buffer I (10 mM imidazole, 100 mM KCl, 1 mM CaCl2, 1 mM MgCl2,
pH 7.0) was calibrated by determining the elution volume
(Ve) of a set of standard proteins of known Stoke's radii: human fibrinogen (107 Å), bovine thyroglobulin (85 Å), horse spleen ferritin (61 Å), bovine liver catalase (52.2 Å),
rabbit muscle aldolase (48.1Å), bovine serum albumin (35.5 Å),
ovalbumin (30.5 Å), bovine pancreas chymotrypsinogen A (20.9 Å), and
bovine pancreas ribonuclease A (16.4 Å). The void volume (Vo) was determined using blue dextran. The
Stoke's radius of TP1 was determined from the calibration curve
obtained by the linear fitting of Stoke's radius versus
( Circular Dichroism--
CD spectra of TP1 and
poly-L-proline (Mr 5000; Sigma
P-2254) in 10 mM potassium phosphate buffer, pH 7.0, were
recorded on a JASCO-J-600 spectropolarimeter (Easton, MD) at seven
temperatures (2, 10, 20, 30, 40, 50, and 70 °C) under a constant
nitrogen flow at 9 liters/min to flush away oxygen that absorbs below
200 nm. The instrument was calibrated with a 0.06% aqueous solution of ammonium D-10-camphosulfonate in the near UV region
and with a 0.015% aqueous solution of D-( ELISA Protein Binding Assays--
Purified TP1 (220 µg/ml in
20 mM sodium phosphate, 1 mM EDTA, 150 mM NaCl, pH 7.0) was absorbed onto microtiter plates
overnight at 4 °C, washed once with TBS-T (10 mM
Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween 20), and
blocked with 0.02% (w/v) bovine serum albumin in TBS-T for 1 h at
37 °C, followed by incubation with actin and nebulin fragments (NA3,
NA4, NC17, ND8, ND66) in buffer I (10 mM imidazole, 150 mM KCl, 1 mM CaCl2, 2 mM MgCl2, pH 7.0) at a concentration ranging
from 0.7 to 50 µg/ml for 2 h at 37 °C. After washing plates
three times with TBS-T, mouse monoclonal antibodies against actin
(JL20), NA3 (N109), NA4 (N103), NC17 (N107), ND8 (N113), and ND66
(N113) were incubated for 1 h at 37 °C in 0.2% bovine serum
albumin-TBS-T. Plates were washed three times with TBS-T and then
incubated with a peroxidase-conjugated rabbit anti-mouse antibody for
1 h. at 37 °C in bovine serum albumin-TBS-T, followed by five
washes with TBS-T. Color was developed for 20 min at 25 °C by adding
ABTS-H2O2 substrate, 10 µl of
H2O2, 10 ml of 100 mM citrate
buffer, pH 4.2. Absorbance at 405 nm was measured using an ELISA
microtiter plate reader (12, 16).
Surface Plasmon Resonance Assays--
Surface plasmon resonance
assays were done using a real time biosensor, IAsys Manual System
(Affinity Sensors, Cambridge, UK). TP1 was attached to
caboxymethylated dextran on the cuvette surface via EDC/NHS.
The cuvette was activated with 0.4 M EDC, 0.1 M
NHS in water for 8 min, washed twice with phosphate-buffered saline with 0.05% Tween 20, followed by a wash with 10 mM
acetate buffer pH 5.0 for 3 min, and incubated with PT1 (0.22 mg/ml in 10 mM acetate buffer, pH 5.0) for 10 min. The cuvette was
washed twice with phosphate-buffered saline with 0.05% Tween 20, followed by 1 M ethylenediamine for 3 min to block excess
activated carboxyl groups. The cuvette was then washed with acetate
buffer (3×), 10 mM HCl, acetate buffer (3×), and finally
PBST (3×). A blank cuvette from the same lot was processed in
parallel, without TP1. The use of ethylenediamine to block excess
carboxyls is essential for the study of the interaction of TP1 with the
highly basic nebulin fragments. The use of hydroxylamine, as
recommended by the manufacturer, led to unacceptably high binding of
basic proteins to the control blank cuvette even at high ionic strength
(200 mM KCl). The introduction of basic groups to the
dextran led to a gel layer that showed no significant electrostatic
binding of NA4 (pI = 9.24) at and above 100 mM KCl
(with response <50 arc seconds).
The binding of nebulin fragments (NA3, NA4, NA29, NC17, and ND66),
actin, troponin, tropomyosin, calmodulin (bovine brain), and S100 Immunogold Localization--
Localization of epitopes of
monoclonal antibodies RT11, RT13, and RT15 in mechanically split rabbit
psoas muscle fibers was done as described previously (18). Briefly, the
split muscle fibers from rabbit psoas muscle in relaxing buffer were
hand-stretched to various degrees and attached to gold single-slot
grid, fixed in 3.7% formaldehyde, labeled with anti-titin
supernatants, and followed by rabbit anti-mouse immunoglobulin and then
protein A gold bead (5 nm) conjugates. The labeled fibers were fixed in glutaraldehyde before embedded in Epon-Araldite, sectioned, and stained
with uranyl acetate and Reynold's lead citrate for observation in an
electron microscope (JEOL 100CX).
Epitope translocation was determined by plotting the center to center
distance from each epitope to the Z line or to the M line as a function
of sarcomere length.
Tandem Repeats of a ~28-Residue PEVK Module in the PEVK Segment
of Titin--
Sequence analysis of a 2.5-kb cDNA clone (5-1-2)
obtained by screening a human fetal muscle cDNA library with
anti-titin antibodies revealed an open reading frame (designated as
hfT11) of 786 residues that is enriched in prolines, glutamates,
valines, and lysines (25% Pro, 16% Glu, 15% Val, 16% Lys)
(GenBankTM accession number AF 321609). A search for
internal sequence homology by matrix plots with MacVector indicates
substantial internal repeats (Fig. 2A). Further analysis
with MACAW to search for sequence homology alignment revealed that the
majority of the sequence near the C-terminal side consists of 18 tandem
repeats of sequence modules of an average of 28 residues (ranging from
25 to 30). The alignment of these modules and nonrepeat sequences in
Fig. 1 showed the consensus of
PE(V/A)PKEVVPEKK(A/V) PVAPPKKPE(V/A)PPVKV. The variability in
length among these modules occurs between the first and second
prolines, between the EKK and PP and between PP and VKV (in
length). Closer examination of these repeats further revealed the
presence of adjoining superrepeats from HRT14-20 (designated as SRA)
and HRT 21-27 (designated as SRB). Within each superrepeat, seven
modules are arranged in the same order of abcdefg types of module. The
N-terminal side of this open reading frame is more variable. Four
modules (HRT1-4) with low similarity are found. The next
four modules, HRT10-13, show little homology with the modules from
HRT14-27, but each module has ~25-35 residues, and three show a PE
at the beginning of the module. These four modules are classified as
nonrepeats. This nonrepeat region is characterized by clusters of
acidic residues (e.g. HRT4) and basic residues (KRRRK in
HRT8). The matrix plot of hfT11 against human soleus PEVK indicates
that hfT11 open reading frame sequence shares significant homology with
the C-terminal side of the PEVK region of human soleus titin (Fig.
2A).
The PEVK region of human fetal skeletal muscle titin sequenced so far
clearly indicates three levels of motifs: repeats of ~28 residues,
superrepeats of seven modules, and nonrepeat. The same research for
internal repeats of human soleus PEVK (residues 0-2174, corresponding
to 5618-7792 of EMBL X90569) revealed the same types of sequence
motifs. The matrix plot in Fig. 2B indicates significant
repeats between residues 300 and 1840 of the PEVK segment of human
soleus titin. Indeed, similar 28-residue repeats are abundant, and
seven-module superrepeats are also present. The balance of the sequence
is nonrepeat and contains clusters of acidic residues (see Refs. 2 and
19). In summary, PEVK of titin appears to be modular and consists
mainly of tandem repeats of a fundamental 28-residue module
interspersed in highly charged nonrepeat regions.
Conformation of PEVK and Polyproline II (PPII) Helix--
The
presence of the modular structure of PEVK, previously unrecognized,
raised the question of whether PEVK may have stable structural folds.
To explore this possibility, we expressed in Escherichia
coli a 469-residue fragment (from 128-597, HRT5 to HRT21,
designated as TP1) that includes both the nonrepeat and part of the
superrepeat of PEVK. To avoid possible interference by expression tags
in protein interaction studies, a nonfusion protein was produced. As
shown in Fig. 3, TP1 was expressed as a
soluble protein in reasonable yield. Due to its low UV absorption (a
single tyrosine residue at position 155), purification and expression
of TP1 were monitored by SDS gel electrophoresis and Western blotting
with a monoclonal anti-titin, RT11. TP1 displayed unusually low SDS gel
mobility and migrated with an apparent mass of 86 kDa. Moreover, TP1
was also difficult to transfer electrophoretically, and most TP1 stayed
behind in the gel (Fig. 3A, post-transfer gel pattern). The
preferential transfer of smaller degradation products of TP1 gave a
smear in Western blots that significantly underestimated the purity of
TP1 (Fig. 3A, RT11 Western blot). The low mobility may
reflect either the lower than normal SDS binding or an extended
conformation or stiffer SDS peptide complex for proline-rich peptides.
For example, a proline-rich protein, calphotin (20), displays a similar
low motility.
Purified TP1 is very soluble in a wide range of aqueous buffers and
ionic strengths. The possible presence of secondary structure in TP1
was investigated by circular dichroism. CD spectra of TP1 display
negative ellipticity with a minimum at 201 nm and a shoulder near 220 nm (Fig. 4D). At first
impression, these are characteristic spectra of polypeptides generally
classified as random coils. Indeed, calculations for secondary
structure content with several software programs indicate negligible
A detailed comparison of CD spectra of polyproline and TP1 strongly
supports this notion. As shown in Fig. 4, A and
B, CD spectra of polyproline show the characteristic strong
negative band at 205 nm and a weak positive band at 229 nm of PPII
helices (21). Upon heating from 2 to 70 °C, both bands undergo
incremental decrease in magnitude (Fig. 4A,
inset), indicating a loss of PPII helical content at higher
temperature. This series of CD spectra (Fig. 4A) displays an
isodichroic point at 215 nm, indicating an equilibrium of two major
populations of conformations. Another unique characteristic of PII is
its response to urea and guanidinium chloride treatment (23). These
chaotropic agents that commonly cause unfolding and loss of most
secondary and tertiary structures in fact increase the helical content
of PPII (23). As shown in Fig. 4B, urea treatment up to 8 M causes an increase in magnitude of bands at 205 and 229 nm, confirming an earlier report of the enhancement effect of urea on
PPII content (23). The presence of an isodichroic point at 218 nm
suggests again two populations of equilibrating conformations. High
concentrations of urea progressively obscure the spectra below 205 nm
and somewhat affect the accuracy of the isodichroic point (Fig.
4B, inset). The third characteristic behavior of
PPII is the nearly linear response to thermal titration, without the
sharp, sigmodial transition commonly observed for
The CD of TP1 and its responses to thermal and urea titration bear
striking resemblance to those of polyproline under identical conditions
(Figs. 4, D-F). The strong negative band at 200 nm is
slightly blue-shifted from the 205-nm band of polyproline. The negative
shoulder at 220 nm may be derived from the same transition as the
229-nm (positive) band of polyproline (Fig. 4, D and
E). Upon heating from 2 to 70 °C, a significant decrease
of the magnitude of both bands of TP1 occurs (Fig. 4E,
inset), showing the loss of stable folds. The presence of an
isodichroic point near 210 nm between 2 and 50 °C suggests that at
least two conformations are in equilibrium between below 50 °C (Fig.
4D). The addition of urea up to 8 M causes
progressive decrease in the 200-nm band and an increase in the shoulder
at 220 nm, with an isodichroic point at 210 nm (Fig. 4E),
consistent with the increase of PPII at higher concentration of urea.
Thermal titration of TP1 at 201 nm reveals a linear response from 20 to
70 °C, with a slight hint of a kink at 38 °C. Upon cooling, the
CD returns to that observed before heating, with no sign of hysteresis
(data not shown).
Based on the similarity of the CD spectra of TP1 to PPII and the unique
and characteristic responses of the CD spectra of TP1 and PPII to urea
and heat treatment, we suggest that TP1 contain a significant extent of
ordered structures that resembles PPII helices. The absence of
sharp, cooperative transition in thermal or urea titration (24)
suggests that TP1 has an open structure within which the PPII helices
are likely to be dispersed in an open polypeptide with very little long
range interactions. Assuming that PPII is the only ordered structure
that contributes to CD spectra, it can be estimated from residue
ellipticity values at 210 nm that PPII content in TP1 at ~13% of the
residues in TP1 is in the PPII conformation at 20 °C. The PPII
content decreases gradually with rising temperature (~7% at
70 °C) and is slightly enhanced by high concentration of urea
(~15% in 8 M urea). Further conformational analysis is
required to evaluate or rule out the possible presence of other ordered
structures in TP1.
Support of the open structure of TP1 came from the determination of its
Stoke's radius by gel filtration. The value of 85 Å (Fig.
5) is similar to that of thyroglobulin
(260 kDa) and is ~1.4 times larger that of a globular protein at 52 kDa. This value clearly indicates that TP1 is a highly asymmetric
molecule. Taken together, these data suggest that PT1 is an elongated
and open polypeptide with multiple PPII helices. Whether the 28-residue PEVK module is the basic PPII folding unit remains to be established by
higher resolution analysis (25).
Reactivity Profile of TP1 with Proteins of Thin Filaments--
A
survey for potential interaction of TP1 with components of thin
filament (actin, tropomyosin, troponin, and nebulin fragments (NA3,
NA4, NA29, NC17, ND8, and ND66) titin motif I, and calmodulin) was done
with both solid phase ELISA and plasmon resonance-based biosensor. As
shown by ELISA (Fig. 6), TP1 on the
plastic surface binds actin, NA4, and NC17 with moderate affinity
(Kd ~1-5 µM). On the other hand,
tropomyosin, troponin, three nebulin fragments (NA3, NA29, ND66), and
calmodulin showed very little affinity toward TP1.
The reactivity of TP1 was investigated further with the surface plasmon
resonance technique, which allows interaction of untagged proteins to a
hydrophilic dextran surface-bound one to be observed at real time.
Technically, due to the high affinity of the basic nebulin fragments
toward the carboxylated surface layer, it was necessary, following
protein coupling, to neutralize the excess anionic charges by a diamino
compounds to lower background binding. As shown in Fig. 6B,
there was no binding of actin to TP1 in buffers near physiological
ionic strength. It is conceivable that the slow diffusion of F-actin
into the sensor's dextran gel layer may have hampered interaction. Two
nebulin fragments, NA4 and NC17, bind to TP1 rapidly, and nebulin
fragments NA3, NA29, tropomyosin, troponin, and titin motif I show no
affinity under the same condition (Fig. 6B). To test
potential for oligomerization of PEVK, binding of solution phase TP1
was also performed. No interaction was observed (data not shown).
The interaction of TP1 with human nebulin fragments confirms the ELISA
data and suggests that PEVK may be involved in the interfilament
interaction with thin filaments. To search for potential modulators for
such interaction, we tested calmodulin/Ca2+, which has been
shown to dissociate nebulin from either actin or myosin (26). As shown
in Fig. 7A, the binding of NA4
to TP1 was dissociated completely by 7 µM calmodulin at
pCa 3. Premixing NA4 and calmodulin also abolished binding. The
addition of EGTA to the mixture to lower pCa to 8-9 immediately
induced the binding of NA4 to TP1. As controls, we found that in the
absence of calmodulin, NA4 binds to TP1 more effectively at pCa 8 than
at pCa 3 (Fig. 7B). Since TP1 does not bind to calmodulin at
either pCa 3 or 8, the data indicate that its dissociative effect of
calmodulin at high calcium is mediated mainly through the binding of
Ca2+/calmodulin to NA4, perhaps by competing with PEVK for
the same binding site on NA4. A direct effect of calcium on either
nebulin or TP1 may also contribute to the lowered binding of NA4 to TP1 at pCa 3. In parallel experiments, we observed a similar dissociative effect of S100
In summary, NA4 binds to TP1 at pCa 8 with moderate, micromolar
affinity. Their binding is weakened by either raising calcium to pCa 3 or by the presence of calmodulin (data not shown). This binding is
completely abolished at pCa 3 in the presence of calmodulin. The
S100 Elastic Stretch Response of PEVK in the Sarcomere--
The
reactivity of a panel of four monoclonal antibodies was tested against
TP1. Only RT11 reacts specifically by ELISA (Fig. 8A). The same results were
also observed by plasmon resonance assays (data not shown). This
epitope is localized in rabbit psoas skeletal muscle by immunogold
labeling at the electron microscopy resolution to a single band
approximately at 0.35 µm from the Z line in the I band of a resting
sarcomere at 2.3 µm (Fig. 8B). The antibody to Z line
distance increases linearly from 0.35 to 0.54 µm when the
sarcomere was stretched from 2.3 to 3.5 µm. This stretch behavior, as
illustrated in the linear plots of Ab to Z line (or Ab to M line)
distance versus sarcomere length (Fig. 8C), is
typical of an elastic response, since the epitope moves away from both
the Z line and the M line proportionally. In contrast, an A band titin
epitope, RT15, was stationary and maintained its position relative to
the M line (Fig. 8C). In a parallel experiment, the epitope
location of another monoclonal antibody RT13 was also measured and
found to be indistinguishable from the location of T11 epitope (8).
Since the exact epitope sequences of RT11 and RT13 remain unknown, it
is likely that both antibodies are directed to an adjacent region of
the PEVK segment of rabbit psaos titin, despite the fact that only RT11
reacts with human fetal titin PEVK.
Modular Structure of PEVK Segment--
The extension of elastic
titin filaments in the I-band is thought to be responsible for the
generation of passive tension of the sarcomere skeletal and cardiac
muscle sarcomeres. Comparison of passive tension and sarcomere length
curves of several skeletal muscles that express several different titin
size isoforms led us to propose that the segmental extension of
different lengths of I band titin accounts for the variation of
sarcomere elasticity of these muscles (3, 8). It is now known that the
I band titin consists of two structural motifs: tandem Ig domains and the PEVK region. Recent mechanical studies revealed that both elements
contributed to passive tension. At low tension, the tandem Ig domains
extend up to 3-4-fold without unfolding. At higher stretch, PEVK
extends and develops higher tension. The two springs work in series to
provide a broad range of forces (29, 30). PEVK is thought to be in a
random coil or permanently unfolded conformation, and its mechanical
behavior has been modeled as an entropic spring (10, 31, 32).
Our analysis of human fetal titin PEVK sequence provides the first
evidence of modular motifs consisting of a fundamental module that
averages 28 residues. In addition, a higher order superrepeat
consisting of seven PEVK modules is also evident. Examination of PEVK
sequences of the 1800 residues of human and rabbit soleus muscles of
Labeit and Kolmerer also reveals the same type of sequence modules and
superrepeats throughout the entire PEVK region. Such motifs, however,
have escaped the attention of these authors in their sequence analysis
(1). Recently, Greaser (19) reported the modular repeats of PEVK while
this manuscript was in preparation. The modular motif of PEVK region is
now also evident in Drosophila titin, where an exact repeat of 100 amino acid residues has been reported (34). The distinct length
and sequence of the modules of human and fly PEVK segment are in
contrast to the similarity of size of the immunoglobulin and
fibronectin domains for the rest of the titin molecule. This comparison
indicates that the distinctly longer PEVK module in fly may meet
specific mechanical demands of titin in invertebrates.
PPII Helices in PEVK--
Conformational studies of polyproline
and TP1 by CD indicate that PEVK in aqueous solution has open but
somewhat folded conformations, most likely a chain of PPII-like helices
with flexible joints. No conventional secondary structure such as
PEVK and Interfilament Adhesion--
Studies with solid phase and
surface plasmon resonance assays indicate that TP1 interacts with actin
and cloned nebulin fragments. Interestingly, NA4/PEVK interaction is
weakened by high calcium and is, furthermore, abolished by Ca/CaM or
Ca/S100 in high calcium. These data suggest that the PEVK region of
titin may be involved also in interfilament association with thin
filaments in a Ca2+ and Ca2+/CaM- or
Ca2+/S100-sensitive manner. This adhesion in turn may
modulate titin extensibility. The lack of interaction of TP1 with some
of the nebulin fragments, however, is puzzling. Fragments NA3, NA29, and ND66 either lack binding sites for PEVK or are not folded or
dispersed properly for molecular contacts. The lack of binding to
tropomyosin and troponin suggests that the titin/thin filament adhesion
is limited to actin and certain loci along the nebulin filament. Since
NA4 and NC17 are normally located in the overlapping region of the A
band in resting sarcomeres (18, 28), PEVK interaction would only occur
when sarcomeres are stretched extensively for PEVK and NA4 to make
contact. Whether the extensibility and the modular motif of PEVK play a
role in the strength and sites of interaction with thin filaments are
intriguing questions for future pursuits. It is tempting to speculate
that the stretching of the PEVK segment might affect its avidity to
interacting protein by changing accessibility and orientation. In this
connection, the seven-module superrepeat structure is intriguing. The
7-fold repeat may match the seven-module nebulin superrepeat (12, 27) and the periodicity of the troponin actin filaments. A triple-stranded interaction between PEVK, nebulin, and actin might occur when titin is
stretched to straighten the PEVK for a zipper-like adhesion with the
thin filaments at the appropriate sarcomere length.
The dissociate effects of Ca2+/calmodulin and
Ca2+/S100 on PEVK/nebulin interaction suggest that this
interfilament connectivity may be modulated by calcium via calmodulin
or S100 type sensor proteins. This modulation is in line with the
observation that these molecules also dissociate nebulin from myosin
and weaken their affinity to actin (26). Thus, calmodulin/S100 sensor
proteins may untether simultaneously adhesion of nebulin to both titin and myosin filaments in the sarcomere during muscle activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix and
-sheet types of secondary structure. Protein
interaction studies with solid phase assays reveal modest micromolar
range interactions between TP1 and nebulin and actin. Our data suggest
that the PEVK region may serve multiple functions. It may serve as an
entropic spring of a chain of structural folds. The PEVK region also
may be a site of interaction with other myofilament proteins to form
interfilament connectivity in the sarcomere.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt11 human fetal skeletal muscle library was screened
by Western blot with a goat anti-rabbit skeletal muscle titin (goat 812) that has been absorbed with E. coli blots and
supplemented with a mixture of monoclonal antibodies (RT10, -11, -13, and -15) (8). One clone, 5-1-2, containing a 2.5-kb open reading frame (designated as hfT11; GenBankTM accession number AF
321609) was subcloned into Bluescript by ECoR1 digestion and
ligation. Nested set deletions were produced by exoIII digestion, and
each clone was sequenced by Sequence/T7 polymerase/universal and
reverse primer with the dideoxy method. The 7-deaza reaction was run to
clear up compression. The DNA sequence and deduced amino acid sequence
were analyzed by MacVector and MACAW for homology and repeats (12).
-D-galactopyranoside induction (0.4 mM isopropyl-1-thio-
-D-galactopyranoside when A550 = 0.6-0.8). The bacteria were
collected by centrifugation at 3800 × g for 10 min and
lysed in a French press cell (3 × 1500 p.s.i.) in 45 ml of
lysis buffer (10 mM NaPi, 1 mM EDTA, 0.1 mM DTT, 10 µg/ml leupeptin, 1 mg/ml casein, 1 mM diisopropyl fluorophosphate, 0.5 mM
phenylmethylsulfonyl fluoride, pH 7.0), followed by centrifugation at
12,000 × g for 20 min at 4 °C. The supernatant was
dialyzed overnight against (10 mM NaPi, 1 mM
EDTA, 0.1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, pH 7.0) at 4 °C, clarified by centrifugation at
27,000 × g for 20 min, and loaded to a Whatman
phosphocellulose P-11 column (5.2 × 4.0 cm) equilibrated in the
same buffer at 4 °C. TP1 was eluted between 0.4 and 0.5 M NaCl by a linear gradient (0-1 M NaCl).
Pooled fractions containing TP1 were dialyzed overnight against 20 mM NaPi, 1 mM EDTA, 0.1 mM DTT, pH
7.0, at 4 °C and applied to a Source 15S PE column (Amersham
Pharmacia Biotech) equilibrated in the same buffer at 4 °C. Elution
with a linear NaCl gradient (0-0.3 M) yielded ~95% pure
TP1 between 0.10 and 0.12 M NaCl (15 mg/4-liter culture).
-mercaptoethanol, pH 10.4); nitrocellulose membrane presoaked in
anode buffer 2 (25 mM Tris-Cl, 0.05% SDS, 10% methanol,
10 mM
-mercaptoethanol, pH 10.4); SDS gels presoaked in
the cathode buffer (25 mM Tris-Cl, 0.05% SDS, 10%
methanol, 10 mM
-mercaptoethanol, and 40 mM
amino-N-hexanoic acid, pH 10.4) for 15 s; and four sheets of
filter paper presoaked in the cathode buffer. Transfer of proteins was
performed at 1.5 mA/cm2 of gel for 60 min.
log Kav)1/2.
)-pantolactone
(Aldrich, WI) in the far UV region. A quartz cuvette (Hellma,
Plainview, NY) with a 0.01-cm light path was used. For urea titration
at 20 °C, TP1 (22 mg/ml) or polyproline (2 mg/ml) was dialyzed
against various concentrations of urea (2, 4, 6, and 8 M)
in the same buffer. Concentrated urea solutions were prepared in water
by weight using the density data of Kanahara (15) and then adjusted to
desired concentration with water and 10× concentrated buffer. Thermal denaturation of TP1 (2.2 mg/ml) and polyproline (0.25 mg/ml) was monitored by following the change in CD at 205 nm (TP1) or 201 nm
(polyproline) from 20 to 90 °C at a rate of 50 °C/h using a JASCO
model PTC-348W Peltier type thermoelectric control system and a
demountable 0.01-cm path length rectangular cuvette.
(Sigma) to immobilized TP1 was done at 1 µM in 100 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM imidazole, pH 7.0, at 25 °C. For some solutions, EGTA (1 mM) was present to
lower pCa to 8.0. Protein binding to the gel layer alters the
refractive index profile occurring within the evanescent field and
changes the measured resonance angle of the intensity peak (in arc
seconds). The change in arc seconds is assumed to be proportional to
the amount of bound substance (17). The association kinetics was followed for 10-15 min prior to washing with the interaction buffer to
follow the time course of dissociation. At the end of each experiment,
the cuvette was washed with 10 mM HCl for 1 min and buffer
I (3×) to regenerate the cuvette. All of the data reported here were
obtained on the same cuvette within a 2-day period to facilitate
comparison. Resonance angles of the treated control cuvette were
measured in parallel and subtracted from the corresponding values
obtained with the TP1 cuvette.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (98K):
[in a new window]
Fig. 1.
Partial sequence of PEVK segment of human
fetal skeletal muscle titin. A 2.5-kb cDNA clone (hfT11) was
obtained by screening a human fetal muscle library with a mixture of
polyclonal and monoclonal antibodies to titin. The deduced amino acid
sequence of hfT11 indicates that it corresponds to the C-terminal
region of the PEVK segment of titin. The modular motifs of the open
reading frame were revealed by protein analysis tools in MacVector and
MACAW for homology and repeats. The sequence was divided to 28 sections, HRT1-28. The alignment of repeats (R, HRT1-4,
HRT10-13, and HRT14-27) and two superrepeats (SRA,
HRT14-20; SRB, HRT15-27) and nonrepeat (NR,
HRT5-9) is indicated to the right. The consensus sequence
for the PEVK modules is shown below. Prolines are in
green, valines are in black, basic residues (Lys,
Arg) are in blue, and acidic residues (Asp, Glu) are in
red. The enclosed region, TP1 (468 residues,
Mr 51,491) was expressed in E. coli for the
current study.
View larger version (64K):
[in a new window]
Fig. 2.
Modular motifs of human fetal PEVK and human
soleus PEVK. Matrix plots of hfT11 open reading frame against
human soleus PEVK (A) and of human soleus PEVK against
itself (B) reveal the extensive homology and internal
repeats of both sequences. The off diagonal
spots in A indicate that hfT11 shares extensive
homology with the 1500-1850 residues of human soleus PEVK. The
internal repeats of human soleus PEVK are mostly PEVK type modules.
Some off diagonal spots reflect the
extensive Glu residues in some regions of this long PEVK segment
(e.g. near 1200).
View larger version (42K):
[in a new window]
Fig. 3.
Expression and purification of cloned PEVK
fragment, TP1. TP1 was expressed as a soluble protein in E. coli. The bacteria were lysed in 10 mM NaPi, 1 mM EDTA, 0.1 mM DTT, 10 µg/ml leupeptin, 1 mg/ml casein, 1 mM diisopropyl fluorophosphate, 0.5 mM phenylmethylsulfonyl fluoride, pH 7.0, and clarified for
PC cellulose chromatography (A). TP1 was eluted between 0.4 and 0.5 M NaCl by a linear gradient (0-1 M
NaCl). Pooled fractions were then applied to a DEAE-Sepharose column
(20 mM NaPi, 1 mM EDTA, 0.1 mM DTT,
pH 7.0) (B). TP1 was eluted between 0.15 and 0.20 M NaCl. Yield was 15 mg per 4-liter culture. Note that TP1
migrated abnormally in SDS gels, with an apparent molecular mass of 86 kDa. Immunoblots of column fractions with monoclonal anti-titin RT11
were used to monitor purification and degradation. TP1 resisted
electroblotting, and a majority of TP1 stayed behind in the gel after
transfer (post-transfer gels for fractions 34-36). It also degraded
rapidly during purification, giving rise to many minor bands below TP1
that were immunoactive to RT11. Nearly all smaller peptides in the
final TP1 preparation are degradation products.
-helix and
-sheet structure (data not shown). However, further CD
studies of TP1 subjected to urea and thermal titration raised doubts
about this interpretation and led us to search for evidence for the
presence of other stable structural features. Given the proline-rich
content of TP1, we considered the possible presence of PPII. Such a
left-handed helix, containing three trans-proline residues
per turn, is found in polyproline in aqueous solution (21, 25) and is
also present as short stretches in many globular proteins (22).
View larger version (51K):
[in a new window]
Fig. 4.
Circular dichroism and urea-thermal titration
of poly-L-proline and TP1. CD spectra were recorded on
a JASCO J-715 spectropolarimeter at 22 °C. Each spectrum was scanned
twice at 5 nm/min with a time constant of 4 s. All samples were in
10 mM KPi, pH 7.0, plus urea as specified. All spectra were
corrected for contributions from buffer and urea. Regions below
200-205 nm were obscured by the absorption of high concentrations of
urea. Molar ellipticity per residue is plotted. A,
polyproline (0.25 mg/ml) at seven temperatures. The changes in
ellipticity at 201, 205, and 229 nm are plotted as an inset.
B, urea titration of polyproline (2 mg/ml) from 0-8
M urea. C, thermal titration of polyproline from
2 to 90 °C. The change in polyproline (0.25 mg/ml) ellipticity at
201 nm from 2 to 90 °C was monitored continuously to reveal subtle
changes of slopes at 28 and 90 °C. D, TP1 (2.2 mg/ml) at
seven temperatures. The changes in ellipticity at 201, 208, and 220 nm
are plotted as an inset. E, urea titration of TP1
(2.2 mg/ml) from 0 to 8 M urea. The region from 200-206 nm
is expanded as the inset. F, Thermal titration of
TP1 from 20 to 90 °C. The change in TP1 (2.2 mg/ml) ellipticity at
201 nm from 20 to 90 °C was monitored continuously to reveal a
subtle change of slope at 38 °C.
-helices,
-sheets, and other stable folds that display cooperative melting. As
shown in Fig. 4C, thermal titration of polyproline from 2 to
90 °C, as monitored continuously at 201 nm, shows a liner response,
with slight change in slopes at 28 and 80 °C. Such a titration curve
suggests the gradual loss of PPII with raised temperature without cooperativity.
View larger version (14K):
[in a new window]
Fig. 5.
Stoke's radius of TP1. The Stoke's
radius of TP1 was determined with a gel filtration column by
interpolating from the calibration curve obtained by the linear
fitting of Stoke's radius versus ( log Kav)1/2 for
a series of proteins with known Stoke's radii; human fibrinogen (107 Å), bovine thyroglobulin (85 Å), horse spleen ferritin (61 Å),
bovine liver catalase (52.2 Å), rabbit muscle aldolase (48.1Å),
bovine serum albumin (35.5 Å), ovalbumin (30.5 Å), bovine pancreas
chymotrypsinogen A (20.9 Å), and bovine pancreas ribonuclease A (16.4 Å) were chromatographed in a Superose 6HR column at a flow rate of
0.25 ml/min. Multiple runs of TP1 were done under the same conditions,
and the Stoke's radius was determined. The calculated Stoke's radius
of TP1 is 85 Å.
View larger version (27K):
[in a new window]
Fig. 6.
Interactions of TP1 with thin filament
proteins. A, ELISA. The interactions of TP1 with actin
and cloned nebulin fragments (NA3, NA4, NC17, ND8, and ND66)
were studied by ELISA. TP1 was attached to a microtiter plate (50 µg/ml in 20 mM NaPi, 1 mM EDTA, 150 mM NaCl, pH 7.0) and incubated with actin and nebulin
fragments (0.7-50 µg/ml in 10 mM imidazole, 150 mM KCl, 1 mM CaCl2, 2 mM MgCl2, pH 7.0) at 25 °C. Binding was
detected with monoclonal antibodies to actin (JLA20), NA3 (N109), NC17
(N107), ND8, and ND66 (N113), followed by peroxidase-labeled secondary
antibody. B, surface plasmon resonance. Interactions of TP1
with thin filament proteins (tropomyosin, troponin, cloned nebulin
fragments (NA3, NA4, NC17, and NA29), and titin motif I) were studied
at 25 °C by IAsys assays, a cuvette-based surface plasmon resonance
for real time interaction. TP1 was attached to the dextran surface as
described under "Experimental Procedures." Each protein was diluted
to 1 µM in 100 mM KCl, 1 mM
CaCl2, 10 mM imidazole, pH 7.0. The interaction
is indicated by the increase of signals (in arc seconds) in real
time.
, a calcium sensor protein that is analogous to calmodulin. As shown in Fig. 7C, S100
diminished
binding of NA4 to TP1 at pCa 8. The interaction is completely abolished
by S100
at pCa 3.
View larger version (23K):
[in a new window]
Fig. 7.
Ca2+/CaM and
Ca2+/S100 sensitivity of NA4/TP1 interaction. IAsys
cuvette system for surface plasmon resonance was used to study real
time interaction. A, Ca2+/CaM sensitivity. TP1
was immobilized to the cuvette and incubated for 7 min with 1 µM nebulin fragment NA4 and 7 µM calmodulin
in 10 mM imidazole, 100 mM KCl, 1 mM CaCl2, pH 7.0. EGTA was subsequently added
to a final concentration of 4 mM, followed by buffer wash.
The sharp swings in signals during the change of solutions
(dotted lines) reflect the refractive index
difference of buffers and are not due to the binding/unbinding of the
proteins. B, calcium sensitivity. The interaction of NA4
with TP1 was studied at two calcium concentrations (pCa 8 and pCa 3) in
10 mM imidazole, 150 mM KCl, pH 7.0. C, Ca2+/S100 sensitivity. The interaction of NA4
(1 µM in 10 mM imidazole, 100 mM
KCl, 1 mM CaCl2, pH 7.0) with surface-bound TP1
at pCa 3 was greatly diminished by 1 µM S100 at pCa 8 (by
the addition of 1 mM EGTA) and completely abolished by 1 µM S100 at pCa 3.
has a similar effect, indicating a general response of
NA4/TP1 interaction to this class of calcium sensor proteins.
View larger version (44K):
[in a new window]
Fig. 8.
Immunolocalization and epitope response
curves of RT11 in the PEVK segment of rabbit psoas muscle.
A, immunoreactivity of TP1. A panel of four anti-titin
monoclonal antibodies (RT10, -11, -13, and -15) was tested for
interaction with microtiter plate-bound TP1 by ELISA. Only RT11 reacted
specifically. B, immunogold localization of RT11. The
epitopes for RT11, -13, and 15 are localized by immunogold labeling.
Each gave rise to one band in the sarcomere. C, elastic
stretch response of RT11. Plots of the distance of the antibody to the
Z line and antibody to the M line against the sarcomere length are
linear and indicate that both RT11 and RT13 are in an elastic PEVK
region of rabbit psoas skeletal muscle sarcomeres.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix or
-sheet is evident in TP1. Thermal titration causes
gradual and reversible loss of PPII helices in both polyproline and TP1
without any evidence of sigmoid shaped cooperative melting commonly
observed for compact protein domains. Urea titration causes both
polyproline and TP1 to increase their helical content. This
counter-intuitive behavior of PPII results from the fact that this
left-handed helix of trans-prolines does not form main chain
hydrogen bonding as in
-helices or
-sheets. The chaotropic effect
of urea on water structure thus has no detrimental effect on PPII. The
enhancement of PPII content may be due to the direct binding of urea to
the peptides (23). We suggest that in aqueous solution PEVK contains
multiple PPII helices in equilibrium. The chain is likely to be
flexible and would make a significant entropic contribution to
elasticity. On the other hand, the "random coil" or "completely
unfolded" depiction of PEVK in the recent literature (1, 10, 33) is
inadequate and perhaps misleading, since it ignores the possibility of
ordered structure and stable folds. Further evaluation of these
specific conformations and factors, which influence the folding of PPII and chain flexibility, is likely to facilitate understanding of the
generation and modulation of titin elasticity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Wan Li for confirming the sequence of TP1 and Drs. Kan Ma, Albert Jin, Jeff Forbes, and Andrea Sinz for stimulating discussions.
![]() |
FOOTNOTES |
---|
* Preliminary reports of these findings have been presented in meeting abstracts (G. Gutierrez-Cruz, A. H. Van Heerden, and K. Wang (1997) Biophys. J. 72, 279 (abstr.); G. Gutierrez-Cruz, A. H. Van Heerden, and K. Wang (1998) Biophys. J. 74, 349 (abstr.)) at the 41st and 42nd annual meetings of the Biophysical Society. This work was supported in part by National Institutes of Health Grant AR 45315 (to K. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF 321609.
¶ To whom correspondence should be addressed: Bldg. 6, Rm. 401, Laboratory of Physical Biology, NIAMS, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-4097; Fax: 301-402-8566; E-mail: wangk@exchange.nih.gov.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M008851200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: kb, kilobase pair; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; PPII, polyproline II; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; N-hydroxysulfosuccinimide..
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Labeit, S., and Kolmerer, B. (1995) Science 270, 293-296[Abstract] |
2. |
Freiburg, A.,
Trombitas, K.,
Hell, W.,
Cazorla, O.,
Fougerousse, F.,
Centner, T.,
Kolmerer, B.,
Witt, C.,
Beckmann, J. S.,
Gregorio, C. C.,
Granzier, H.,
and Labeit, S.
(2000)
Circ. Res.
86,
1114-1121 |
3. | Wang, K., McCarter, R., Wright, J., Beverly, J., and Ramirez-Mitchell, R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7101-7105[Abstract] |
4. | Pastore, A., Politou, A. S., and Thomas, D. J. (1996) J. Muscle Res. Cell Motil. 17, 114-115 |
5. | MuhleGoll, C., Nilges, M., and Pastore, A. (1997) J. Biomol. NMR 9, 2-10[Medline] [Order article via Infotrieve] |
6. |
Kellermayer, M. S. Z.,
Smith, S. B.,
Granzier, H. L.,
and Bustamante, C.
(1997)
Science
276,
1112-1116 |
7. | Linke, W. A., Ivemeyer, M., Olivieri, N., Kolmerer, B., Ruegg, J. C., and Labeit, S. (1996) J. Mol. Biol. 261, 62-71[CrossRef][Medline] [Order article via Infotrieve] |
8. | Wang, K., McCarter, R., Wright, J., Beverly, J., and Ramirez-Mitchell, R. (1993) Biophys. J. 64, 1161-1177[Abstract] |
9. | Granzier, H. L. M., and Wang, K. (1993) Biophys. J. 65, 2141-2159[Abstract] |
10. |
Linke, W. A.,
and Granzier, H.
(1998)
Biophys. J.
75,
2613-2614 |
11. | Politou, A. S., Gautel, M., Pfuhl, M., Labeit, S., and Pastore, A. (1994) Biochemistry 33, 4730-4737[Medline] [Order article via Infotrieve] |
12. |
Wang, K.,
Knipfer, M.,
Huang, Q. Q.,
VanHeerden, A.,
Gutierrez, G.,
Quian, X.,
and Stedman, H.
(1996)
J. Biol. Chem.
271,
4304-4114 |
13. | Kyhse-Andersen, J. (1984) J. Biochem. Biophys. Methods 10, 203-209[CrossRef][Medline] [Order article via Infotrieve] |
14. | Laurent, T. C., and Killander, J. (1964) J. Chromatogr. 14, 317-330[CrossRef] |
15. |
Kawahara, K.,
and Tanford, C.
(1966)
J. Biol. Chem.
241,
3228-3232 |
16. |
Jin, J. P.,
and Wang, K.
(1991)
J. Biol. Chem.
266,
21215-21223 |
17. | Stenberg, E., Persson, B., Roos, H., and Urbaniczky, C. (1991) J. Colloid Interface Sci. 143, 513-526 |
18. | Wright, J., Huang, Q. Q., and Wang, K. (1993) J. Muscle Res. Cell Motil. 14, 476-483[Medline] [Order article via Infotrieve] |
19. | Greaser, M. L. (2000) Biophys. J. 78, 18A |
20. | Martin, J. H., Benzer, S., Rudnicka, M., and Miller, C. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1531-1535[Abstract] |
21. | Bhatnagar, R. S., and Gough, C. A. (1996) in Circular Dichroism and Conformational Analysis of Biomolecules (Fasman, G. D., ed) , pp. 183-200, Plenum Press, New York |
22. | Adzhubei, A. A., and Sternberg, M. J. (1993) J. Mol. Biol. 229, 472-493[CrossRef][Medline] [Order article via Infotrieve] |
23. | Tiffany, M. L., and Krimm, S. (1973) Biopolymers 12, 575-587 |
24. | Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 211-222[Medline] [Order article via Infotrieve] |
25. | Sreerama, N., and Woody, R. W. (1994) Biochemistry 33, 10022-10025[Medline] [Order article via Infotrieve] |
26. | Root, D. D., and Wang, K. (1994) Biochemistry 33, 12581-12591[Medline] [Order article via Infotrieve] |
27. | Labeit, S., and Kolmerer, B. (1995) J. Mol. Biol. 248, 308-315[CrossRef][Medline] [Order article via Infotrieve] |
28. | Kruger, M., Wright, J., and Wang, K. (1991) J. Cell Biol. 115, 97-107[Abstract] |
29. | Trombitas, K., Greaser, M., Labeit, S., Jin, J. P., and Granzier, H. (1999) Biophys. J. 76, A31 |
30. |
Trombitas, K.,
Greaser, M.,
Labeit, S.,
Jin, J. P.,
Kellermayer, M.,
Helmes, M.,
and Granzier, H.
(1998)
J. Cell Biol.
140,
853-859 |
31. | Trombitas, K., Greaser, M., French, G., and Granzier, H. (1998) J. Struct. Biol. 122, 188-196[CrossRef][Medline] [Order article via Infotrieve] |
32. | Linke, W. A., and Minajeva, A. (2000) Biophys. J. 78, 229A |
33. |
Linke, W. A.,
Ivemeyer, M.,
Mundel, P.,
Stockmeier, M. R.,
and Kolmerer, B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8052-8057 |
34. |
Machado, C.,
Sunkel, C. E.,
and Andrew, D. J.
(1998)
J. Cell Biol.
141,
321-333 |