* Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman,
Washington 99164-6520; Muscle Biology Laboratory, University of Wisconsin, Madison, Wisconsin 53706-1181; § European
Molecular Biology Laboratory, 69012 Heidelberg, Germany; and
Department of Physiology and Biophysics, Case Western
Reserve University, Cleveland, Ohio 44106-4970
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Abstract |
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Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293-296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (~2.0 µm) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from ~2.0 to 2.7 µm, the "contracted" tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond ~2.7 µm, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112-1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force-SL relation that is not achievable with a single elastic segment.
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Introduction |
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TITIN is a giant filamentous protein that, in addition
to the thin and thick filaments, constitutes the third
myofilament system of striated muscle. In the sarcomere, titin molecules span the entire 1-2-µm distance
from the Z-line to the M-line. Previous studies have revealed that the A-band region of the molecule is rendered
inextensible due to its tight association with the thick filament, whereas the I-band region behaves elastically as the sarcomere undergoes changes in length. The elastic properties of the I-band region of titin are primarily responsible for the passive force that is generated when unactivated (i.e., passive) muscle is stretched. Passive force is
present in actively contracting muscle as well, where it
helps maintain the structural integrity of the sarcomere
and thereby ensures efficient muscle contraction. (For recent reviews and original citations see Fürst and Gautel, 1995; Trinick, 1996
; Wang, 1996
; Labeit et al., 1997
;
Maruyama, 1997
.) The recently elucidated primary structure of human soleus titin (Labeit and Kolmerer, 1995
) indicates that in the I-band titin is composed mainly of two
types of segments: tandem Ig segments, consisting of serially linked Ig-like domains, and the PEVK segment that
has a unique sequence (70% of its residues are P-proline, E-glutamate, V-valine, and K-lysine). The PEVK and tandem Ig segments have been suggested to act as low- and
high-stiffness segments of the molecule, respectively (Labeit and Kolmerer, 1995
).
In earlier studies of the elastic behavior of titin, Trombitás et al. (1995) and Granzier et al. (1996)
found that in
slack sarcomeres (where passive force is zero) the elastic
I-band portion of titin is not straight, rather, it is in a contracted state. Passive force was proposed to be determined
by the entropic force arising from the straightening of the
I-band region of titin during modest stretch and by the unraveling (denaturation) of domains at high degrees of sarcomere extension. Subsequently, Gautel and Goulding
(1996)
and Linke et al. (1996)
investigated whether extension of the elastic I-band segment occurs uniformly along
the elastic segment. Using immunofluorescence, Linke et al.
(1996)
followed the location of an antibody (N2A) that labels the unique sequence NH2-terminal of the PEVK segment. The titin segment between the N2A epitope and the
Z-line extended predominantly during small amplitude
stretch. However, upon moderate to extreme stretch extension of the segment between the N2A epitope and the
A-band (PEVK segment and ~25 Ig domains) became
predominant. Gautel and Goulding (1996)
used an antibody (MG1) that labels titin some distance NH2-terminal of the PEVK segment and, by using immunoelectron microscopy, monitored the distances from the epitope to the
Z-line and to the A-band as a function of sarcomere length
(SL)1. The titin segment between the MG1 epitope and
the Z-line (containing Ig domains) was found to continuously extend with sarcomere stretch, albeit with a decreased rate in sarcomeres longer than ~2.8 µm. The segment between the MG1 epitope and the A-band (containing both the PEVK segment and ~25 Ig-like domains) extended linearly as sarcomeres were stretched along a wide
SL range. Gautel and Goulding (1996)
and Linke et al.
(1996)
explained the observations with a model in which
the tandem Ig and PEVK segments extend sequentially.
Recent experiments with single molecules have yielded
new insights into the elastic properties of titin (Kellermayer et al., 1997; Rief et al., 1997
; Tskhovrebova and
Trinick, 1997
; Tskhovrebova et al., 1997
). The force versus
extension curves of the titin molecule measured with laser
tweezers revealed that at small to moderate extensions the
wormlike chain (WLC) behavior of an unfolded region
dominates the behavior of the entire molecule (Kellermayer et al., 1997
). The unfolded region may contain the
PEVK segment, because the preponderance of proline
residues and charge clusters along the PEVK sequence
may prevent the formation of stable tertiary structure
folds (Labeit and Kolmerer, 1995
), suggesting that the PEVK behaves as a denatured polypeptide. Upon stretching the single titin molecule with high enough forces and
with appropriately slow rates, unfolding of globular domains occurred (Kellermayer et al., 1997
). To test whether
the PEVK segment behaves as unfolded protein in situ
and whether unfolding of Ig domains takes place under
physiological conditions, the behavior of tandem Ig and PEVK segments must be precisely followed during passive
stretching of the sarcomere.
In this work sequence-specific antibodies were used to
mark the boundaries of the PEVK segment, making it possible for the first time to follow its extension as sarcomeres
were stretched. In addition, we also marked the tandem Ig
segments and studied their extensible behavior. As the
number of residues in the PEVK segment and the number
of Ig-like domains of the tandem Ig segments varies in
titins from different muscles we used human soleus muscle whose titin has been sequenced (Labeit and Kolmerer,
1995). This allowed us to calculate the contour length of
the unfolded PEVK segment and also to convert the measured extensions of PEVK and tandem Ig segment to average extension per single amino acid and average extension
per single Ig-like domain, respectively. It was found that
extension of the tandem Ig segments dominated at SLs between slack (~2.0 µm) and ~2.7 µm, whereas PEVK extension dominated at SLs greater than ~2.7 µm. It was
possible to effectively simulate the extension of the tandem Ig and PEVK segments with the WLC model (Bustamante et al., 1994
; Marko and Siggia, 1995
) using the properties of the single titin molecule (Kellermayer et al.,
1997
). According to our analysis, throughout the physiological SL range the Ig-like domains maintain their folded
structure, and the PEVK segment behaves as a permanently unfolded polypeptide. The serial linking of a permanently folded and a permanently unfolded segment in
titin, due to the segments' significantly different bending
rigidities, provides passive muscle with a unique mechanical stretch response.
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Materials and Methods |
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Fiber Preparations and Mechanics
Single fibers were dissected from human soleus muscle (biopsies obtained
in accordance with protocol "Role of titin in human muscle tissue 2,"
Washington State University) and were both chemically and mechanically
skinned. Fibers were kept continuously in relaxing solution that contained
high levels of protease inhibitors (for example see Granzier and Irving,
1995), and were used within 48 h of harvest. SL was obtained by using laser diffraction. To measure the passive force-SL relation, relaxed fibers
were slowly stretched (35 nm/s per sarcomere) from their slack SL to a
predetermined amplitude, followed by a release back to the slack length.
To measure maximal active force, fibers were activated (SL 2.5 mm) with
10
4 M free Ca2+. (For additional technical details see Granzier and Irving, 1995
; Granzier et al., 1996
.)
Immunoelectron Microscopy
Fibers were stretched (~0.25 µm/sarcomere/s) and held for 10 min followed by fixation, immunolabeling, embedding, and sectioning (Trombitás et al., 1991; Granzier et al., 1996
). The examined antibodies and their
binding sites along the sequence of human soleus titin are listed in Fig. 1
A1. The T12 epitope location is based on Sebestyén et al. (1995)
. N2A is
an affinity-purified polyclonal antibody (Linke et al., 1996
) that was raised
against the expressed titin sequence from the N2A splice pathway (base pairs 15607-15957 of the human soleus titin sequence). Antibody 514 is an
affinity-purified polyclonal antibody that was raised against a synthetic
peptide containing COOH-terminal PEVK amino acid residues 4596-
4606 of human cardiac titin. Ti102 is a monoclonal antibody that was
raised against the genetically expressed cardiac titin fragment I47-I48
(Jin, 1995
; Li et al., 1995
). Z-line to epitope distances were measured from
electron micrographs after high-resolution scanning (UMAX, UC-1260)
and digital image processing using custom-written macros for the image
analysis program NIH Image (v. 1.6, Wayne Rasband, National Institutes
of Health). For spatial calibration, the electron microscope's (JEOL-1200 EX; JEOL Ltd., Tokyo, Japan) magnification was used.
|
Calculations
A recent comparison of several entropic elasticity models (Kellermayer et al.,
1997) revealed that the properties of the single titin molecule can be described best with the WLC model. In the present work we assumed that
titin behaves as two WLCs in series: the tandem Ig segment and the
PEVK segment. For a WLC, the external force (F) is related to the fractional extension (z/L) as:
![]() |
(1) |
where A is the persistence length, k is Boltzmann's constant, T is absolute temperature, z is the end-to-end length (extension), and L is the contour length (equal to the end-to-end length of the chain stretched with infinite force; Bustamante et al., 1994
; Marko and Siggia, 1995
). The contour
length of the fully extended PEVK segment is ~820 nm, calculated from
its 2,174 residues (Labeit and Kolmerer, 1995
) and a maximal residue
spacing of ~0.38 nm (Cantor and Schimmel, 1980
). The contour length of the native tandem Ig segments was estimated by plotting the measured end-to-end length of the tandem Ig segments (Fig. 3) versus passive force
1/2 (passive force measured in single fibers) and extrapolating to infinite force (for example see Kellermayer et al., 1997
). The contour
lengths obtained by this method for the first and second tandem Ig segments (Fig. 1 A1) were 340 and 140 nm, respectively. Because tandem Ig
and PEVK segments are in series, they bear equivalent forces, and the
fractional extension (z/L) of one segment can be calculated as a function
of the fractional extension of the other. From the fractional extensions of
the PEVK and tandem Ig segments the corresponding SLs were calculated using 0.1 mm as the distance from the center of the Z-line to the T12
epitope and 1.6 mm as the A-band width. For additional details see
Granzier et al. (1997)
.
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![]() |
Results |
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Human soleus fibers were stretched and held at constant length for 10 min and subsequently fixed, immunolabeled, and then processed for electron microscopy. Sequence-specific antititin antibodies (T12, N2A, 514, and Ti-102) were used to mark the boundaries of the tandem Ig and PEVK segments. Between the T12 and N2A epitopes lies the first tandem Ig segment, between N2A and 514 epitopes the PEVK segment, and between 514 and Ti102 epitopes the second tandem Ig segment (Fig. 1 A1). From the electron micrographs (examples are shown in Fig. 1), the epitope-to-Z-line distances and the end-to-end lengths of the tandem Ig and PEVK segments were determined as a function of SL.
In short sarcomeres, the four epitopes merged (Fig. 2), indicating that the elastic I-band segment extends from near the T12 epitope to near the Ti102 epitope. In slack sarcomeres (i.e., in sarcomeres where passive force is zero), the end-to-end length of the elastic segment was ~130 nm (Table I). Because this length is much smaller than the ~480-nm contour length of the native tandem Ig segments (see below), the segment between the T12 and Ti102 epitopes is not fully extended in slack sarcomeres; rather, it occupies a contracted state. As sarcomeres were stretched from their slack length of ~2.0 µm to a length of ~2.75 µm, titin extension was confined primarily to the tandem Ig segments. In contrast, upon further stretch, extension occurred primarily within the PEVK segment (Fig. 3). Thus, tandem Ig extension dominates between the slack SL and ~2.75 µm, and PEVK extension dominates at longer lengths.
|
|
To determine the forces required to extend the tandem Ig and PEVK segments, we measured the passive force-SL relation of human soleus muscle fibers. The magnitude of passive force was expressed relative to that of active force established at a SL of ~2.5 µm. Passive force at 2.75 µm SL was 4.1 ± 1.9% (mean ± SD; n = 4) of maximal active force, whereas at 3.75 µm it was 40 ± 10%. Thus, extending the tandem Ig and PEVK segments requires physiologically significant passive force levels.
The behavior of the tandem Ig and PEVK segments in
response to sarcomere stretch was simulated by the WLC
model of entropic elasticity (Bustamante et al., 1994;
Marko and Siggia, 1995
). Experiments on single molecules
have indicated that the PEVK segment may behave as a
permanently unfolded polypeptide, with properties strikingly different from those of the rest of the molecule. In the study by Kellermayer et al. (1997)
, the unfolded region
was found to behave as a WLC with a persistence length (a
measure of the chain's bending rigidity) of ~2 nm. In contrast, the native structure has a persistence length of ~15
nm (Higuchi et al., 1993
). Titin was assumed to behave as
two WLCs in series: the tandem Ig segment and the PEVK
segment. For a WLC, the external force (F) can be calculated from its persistence length (A), and fractional extension (z/L), where (z) is the end-to-end length, and (L) is
the contour length (Bustamante et al., 1994
; Marko and
Siggia, 1995
). Because WLCs in series bear equivalent
forces, the relative fractional extensions of PEVK and tandem Ig segments at a given force level can be calculated.
From the fractional extensions and the contour lengths of
the PEVK and tandem Ig segments, their end-to-end
lengths were calculated as a function of SL (see Materials
and Methods).
A comparison of the simulated and experimental results is shown in Fig. 3. The main difference was that at Sls < 2.8 µm the predicted end-to-end lengths of the PEVK segment were slightly less than measured. This may indicate that, at those Sls, the persistence length of the PEVK segment is slightly larger than the assumed 2 nm. This would result in an increase in the measured end-to-end length of the PEVK segment for a given degree of sarcomere stretch (Materials and Methods). A larger persistence length at Sls < 2.8 µm may be explained if either the entire PEVK segment or subsegments within the PEVK were to assume a favored conformation(s). An alternative explanation is that at Sls < 2.8 µm the PEVK segment interacts weakly with actin (Guttierez, G., A. van Heerden, J. Wright, and K. Wang. Biophys. J. 72:279a (Abstr.)). A possible effect of Ig domain unfolding on the predicted curves was estimated by assuming that a certain number of Ig domains (1, 2, and 4) were preunfolded, and that each behaved as a WLC with persistence length of 2 nm and a contour length of 38 nm [(residue spacing) × (number of residues): 0.38 nm × 100)]. The data were most accurately simulated by assuming that the Ig domains were fully folded (Fig. 3 A).
Tskhovrebova et al. (1997) have measured 4.8 and 0.15 nm
for the persistence length of the folded regions of the titin
molecule and the PEVK segment, respectively. These values are significantly smaller than those measured by
Kellermayer et al. (1997)
and used in the calculations
above. To compare the different reported persistence lengths, we simulated the in situ titin behavior with the
shorter persistence lengths measured by Tskhovrebova et al.
(1997)
as well. The predicted extensions were somewhat
larger than measured for the tandem Ig segments and
smaller than measured for the PEVK segment at SLs less
than ~3.5 µm (Fig. 3). Overall the simulation was less optimal than when using the values reported by Kellermayer et al. (1997)
. Considering the 0.6-220-nm persistence
length range of amino-acid homopolymers (Cantor and
Schimmel, 1980
), the persistence length of the PEVK segment may have been underestimated by Tskhovrebova et
al. (1997)
.
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Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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The elastic region of human soleus titin extends in situ
from near the T12 epitope to near the Ti102 epitope, and
is composed mainly of tandem Ig segments and the PEVK
segment (Figs. 1 and 2). When slack sarcomeres are
stretched, the elastic titin segment does not extend uniformly. Instead, in sarcomeres stretched to a length of
~2.75 mm, titin extension is confined primarily to the tandem Ig segments, whereas upon further stretch, extension occurs primarily within the PEVK segment. These findings
strongly support the sequential extension model of titin
elasticity (Labeit and Kolmerer, 1995; Gautel and Goulding, 1996
; Linke et al., 1996
). To understand how it is possible that titin's distinct segments extend differently, we
simulated the segmental extension measurements obtained here with the mechanical properties of the single
titin molecule (Kellermayer et al., 1997
; Tskhovrebova et al.,
1997
). Modeling the tandem Ig and PEVK segments as serially linked WLC with persistence lengths of 15 and 2 nm,
respectively, resulted in extensions strikingly close to our
measurements (Fig. 3). The simulation provides an explanation for the sequential extension of the two types of segments: significant extension of the high-persistence length
tandem Ig segments occurs at low force whereas extending the low-persistence length PEVK segment requires much
higher forces (Eq. 1).
Modeling the tandem Ig and PEVK segments as serially
linked WLCs results in the most effective simulation when
Ig domain unfolding is assumed to be absent (Fig. 3 A), indicating that in situ, all of the Ig domains occupy the
folded configuration. Assuming that Ig unfolding is indeed
absent, the Ig interdomain spacing at the longest SL studied (4.2 mm) was calculated to be ~4.5 nm (Table I). This
distance can be accommodated by the native structure of
the Ig domains along with their short polypeptide linking
sequence (Improta et al., 1996). Since the in situ SL is unlikely to extend beyond 4.2 µm (Lieber et al., 1994
; Ledvina and Segal, 1995
), Ig unfolding may be absent along
the entire physiological SL range. Stretching single molecules using scanning force microscopy or optical trapping
results in the unfolding of Ig/Fn domains with a rate constant that increases with force (Kellermayer et al., 1997
;
Rief et al., 1997
). Accordingly, a pronounced unfolding was expected to take place during the long time (~10
) allowed by the preparative procedure. The unexpected lack
of unfolding (Fig. 3 A) may result from an interaction between titin and other sarcomeric elements (Granzier et al.,
1997
), which could stabilize Ig domains, and allow muscle
to avoid the energetically costly unfolding/refolding cycle.
In conclusion, the effective simulation of tandem Ig extension that results from assuming permanently folded Ig domains, indicates that Ig unfolding is likely to be absent
throughout the physiological SL range.
The average PEVK residue spacing in the longest sarcomeres studied (4.2 mm) was ~0.33 nm (Table I). This
distance can be accommodated by the PEVK segment if it
is fully unfolded and extended to nearly its maximal length
(Cantor and Schimmel, 1980). Because the simulation of
PEVK extension (see above) assumed WLC behavior without unfolding/refolding transitions, the closeness of
the measured and simulated data (Fig. 3 B) indicates that
the PEVK segment behaves as an unfolded polypeptide at
all SL. Avoiding unfolding/refolding transitions will minimize energy loss (hysteresis) during stretch-release cycles
(Kellermayer et al. 1997
). However, some hysteresis may
still be present. For example, time delays in straightening
of thermal fluctuations along the PEVK segment may give
rise to hysteresis during stretch-release cycles with high
speed (Seifert et al., 1996
). In conclusion, the PEVK segment extends from a contracted state to a straightened
state as sarcomeres are stretched. Like other recently discovered proteins with unfolded but biologically active domains (Daughdrill et al., 1997
; Plaxco and Groß, 1997
),
titin may be a protein for which the unfolded state is functionally important.
The N2A antibody has recently been used to follow the
NH2-terminal end of the PEVK segment (Linke et al.,
1996). This antibody together with the novel antibody specific to the COOH-terminal PEVK region (antibody 514)
made it possible for the first time to study how the entire
PEVK segment extends as the sarcomere is stretched (Fig.
3 B). The Z-line to N2A epitope distances measured here
(Fig. 2) are in general agreement with those obtained on rat soleus myofibrils using immunofluorescence (Linke et al., 1996
), suggesting that soleus titins of different species are conserved. In contrast, different observations were made
on rabbit psoas fibers by Gautel and Goulding (1996)
using an antibody (MG1) that labels near the N2A epitope.
The distance from the Z-line to the MG1 epitope increased by ~180 nm between SLs 2.0 and 2.7 µm, and by
an additional ~100 nm between SLs 2.7 and 3.5 µm. In this
study (Fig. 2), the Z-line to N2A distance remained almost constant at SLs greater than ~2.7 µm. The conflicting results might be caused by differences in the properties of
human soleus and rabbit psoas titins, or by differences in
the experimental protocols used. Our work indicates an
absence of Ig-unfolding in sarcomeres stretched and then
held in that state for ~10
. However, due to the kinetic nature of unfolding (Rief et al., 1997
; Kellermayer et al.,
1997
), Ig domain unfolding may occur if sarcomeres are
held in the stretched state long enough. Thus, in principle,
a long preparation procedure may have caused the increase in Z-line-to-MG1 distances in sarcomeres longer
than ~2.75 µm (Gautel and Goulding, 1996
).
The mechanical functions of titin include passively restoring stretched or shortened sarcomeres to their slack
length (Trombitás et al., 1993, 1997
; Helmes et al., 1996
),
maintaining the central position of A-bands in contracting
sarcomeres (Horowits et al., 1987) and limiting SL inhomogeneity amongst series-coupled sarcomeres (Granzier
and Pollack, 1990
; Granzier et al., 1991
). To perform these
important physiological functions, the relation between titin's force and SL can neither be too shallow (Horowits
et al., 1987; Granzier and Pollack, 1990
; Granzier et al.,
1991
), nor too steep, as this would prevent sarcomeres
from operating over a wide range of lengths, which in
some skeletal muscles ranges from ~2.5 to 3.75 µm (Lieber et al., 1994
). Titin meets these functional requirements
by serially linking two types of segments with strikingly
different elastic properties. Due to their high-persistence length, the tandem Ig segments extend at low forces (Fig.
4, inset) and are unable to provide a physiologically relevant passive force range by themselves. In combination
with the PEVK segment, however, the tandem Ig segments may behave as length regulators that set the SL at
which PEVK-based passive force generation begins (Fig. 4). Conversely, the low-persistence length PEVK segment
requires high forces for moderate extension (Fig. 4, inset)
and is unable to provide a wide SL range with physiologically relevant passive forces by itself. However, in series
with the tandem Ig segments the PEVK segment may
function as a passive force generator (Fig. 4) that develops
intermediate forces (2-20 pN) at a wide range of SLs (2.7-
4.2 µm). In addition, the permanently unfolded state of
the PEVK segment makes repeated stretch and release
possible with minimal energy loss. Thus, the unfolded
PEVK segment of low-persistence length in series with the
folded tandem Ig segment of high-persistence length may
provide an efficient way of developing physiologically useful levels of passive force over a wide SL range.
|
![]() |
Footnotes |
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Received for publication Received for publication 13 November 1997 and in revised form 18 December 1997..
1. Abbreviations used in this paper: SL, sarcomere length; WLC, wormlike chain. ![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Bustamante, C.,
J. Marko,
E. Siggia, and
S. Smith.
1994.
Entropic elasticity of
![]() |
2. | Cantor, C., and P. Schimmel. 1980. The Behavior of Biological Macromolecules. Freeman and Company, San Francisco. 849-1371. |
3. |
Daughdrill, G.,
M. Chadsey,
J. Karlinsey,
K. Hughes, and
F. Dahlquist.
1997.
The C-terminal half of anti-sigma factor, FlgM, becomes structured when
bound to its target, ![]() |
4. | Fürst, D., and M. Gautel. 1995. The anatomy of a molecular giant: how the sarcomeric cytoskeleton is assembled from molecules of the immunoglobulin superfamily. J. Mol. Cell. Cardiol. 27: 951-959 |
5. | Gautel, M., and D. Goulding. 1996. A molecular map of titin/connectin elasticity reveals two different mechanisms acting in series. FEBS Lett. 385: 11-14 |
6. | Granzier, H., and G. Pollack. 1990. The descending limb of the force-sarcomere length relation of the frog revisited. J. Physiol. 421: 595-615 [Abstract]. |
7. | Granzier, H., and T. Irving. 1995. Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments. Biophys. J. 68: 1027-1044 [Abstract]. |
8. |
Granzier, H.,
H. Akster, and
H. ter Keurs.
1991.
Effect of thin filament length
on the force-sarcomere length relation of skeletal muscle.
Am. J. Physiol.
260:
C1060-C1070
|
9. | Granzier, H., M. Helmes, and K. Trombitás. 1996. Nonuniform elasticity of titin in cardiac myocytes: a study using immunoelectron microscopy and cellular mechanics. Biophys. J. 70: 430-442 [Abstract]. |
10. | Granzier, H., M. Kellermayer, M. Helmes, and K. Trombitás. 1997. Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction. Biophys. J. 73: 2043-2053 [Abstract]. |
11. | Helmes, M., K. Trombitás, and H. Granzier. 1996. Titin develops restoring force in rat cardiac myocytes. Circ. Res. 79: 603-611 . |
12. |
Higuchi, H.,
Y. Nakauchi,
K. Maruyama, and
F. Fujime.
1993.
Characterization of ![]() |
13. | Horowits, R., and R. Podolsky. 1987. The positional stability of thick filaments in activated skeletal muscle: evidence for the role of titin filaments. J. Cell Biol 105: 2217-2223 [Abstract]. |
14. | Improta, S., A. Politou, and A. Pastore. 1996. Immunoglobulin-like modules from titin I-band: extensible components of muscle elasticity. Structure. 4: 323-337 |
15. |
Jin, J.-P..
1995.
Cloned rat cardiac titin class I and class II motifs: expression,
purification, characterization, and interaction with F-actin.
J. Biol. Chem.
270:
6908-6916
|
16. |
Kellermayer, M.,
S. Smith,
H. Granzier, and
C. Bustamante.
1997.
Folding- unfolding transitions in single titin molecules characterized with laser tweezers.
Science.
276:
1112-1116
|
17. | Labeit, S., and B. Kolmerer. 1995. Titins, giant proteins in charge of muscle ultrastructure and elasticity. Science. 270: 293-296 [Abstract]. |
18. |
Labeit, S.,
B. Kolmerer, and
W. Linke.
1997.
The giant protein titin. Emerging
roles in physiology and pathophysiology.
Circ. Res.
80:
290-294
|
19. |
Ledvina, M., and
S. Segal.
1995.
Sarcomere length and capillary curvature of rat
hindlimb muscles in vivo.
J. Appl. Physiol.
78:
2047-2051
|
20. | Li, Q., J.-P. Jin, and H. Granzier. 1995. The effect of genetically expressed cardiac titin fragments on in vitro actin motility. Biophys. J. 69: 1508-1518 [Abstract]. |
21. | Lieber, R., G. Loren, and J. Fridén. 1994. In vivo measurement of human wrist extensor muscle sarcomere length changes. J. Neurophysiol. 71: 874-881 [Abstract]. |
22. | Linke, W., M. Ivemeyer, M. Olivieri, B. Kolmerer, J. Rüegg, and S. Labeit. 1996. Towards a molecular understanding of the elasticity of titin. J. Mol. Biol. 261: 62-71 |
23. | Marko, J., and E. Siggia. 1995. Stretching DNA. Macromolecules. 8: 8759-8770 . |
24. |
Maruyama, K..
1997.
Connectin/titin, giant elastic protein of muscle.
FASEB J
11:
341-345
|
25. | Plaxco, K., and M. Groß. 1997. The importance of being unfolded. Nature. 386: 657-659 |
26. |
Rief, M.,
M. Gautel,
F. Oesterhelt,
J. Fernandez, and
H. Gaub.
1997.
Reversible unfolding of individual titin immunoglobulin domains by AFM.
Science.
276:
1109-1112
|
27. |
Sebestyén, M.,
J. Wolff, and
M. Greaser.
1995.
Characterization of a 5.4 kb
cDNA fragment from the Z-line region of rabbit cardiac titin reveals phosphorylation sites for proline-directed kinases.
J. Cell Sci.
108:
3029-3037
|
28. | Seifert, U., W. Wintz, and P. Nelson. 1996. Straightening of thermal fluctuations in semiflexible polymers by applied tension. Phys. Rev. Lett. 77: 5389-5392 . |
29. | Trinick, J.. 1996. Cytoskeleton: titin as scaffold and spring. Curr. Biol. 6: 258-260 |
30. | Trombitás, K., P. Baatsen, M. Kellermayer, and G. Pollack. 1991. Nature and origin of gap filaments in striated muscle. J. Cell Sci. 100: 809-814 [Abstract]. |
31. | Trombitás, G., Pollack, J. Wright, and K. Wang. 1993. Elastic properties of titin filaments demonstrated using a "freeze-break" technique. Cell Motil. Cytoskel. 24: 274-283 |
32. |
Trombitás, K.,
J.-P. Jin, and
H. Granzier.
1995.
The mechanically active domain
of titin in cardiac muscle.
Circ. Res.
77:
856-861
|
33. | Trombitás, K., M. Greaser, and G. Pollack. 1997. Interaction between titin and thin filaments in intact cardiac muscle. J. Muscle Res. Cell. Motil. 18: 345-351 |
34. | Tskhovrebova, L., and J. Trinick. 1997. Direct visualization of extensibility in isolated titin molecules. J. Mol. Biol. 265: 100-106 |
35. | Tskhovrebova, L., J. Trinick, J. Sleep, and R. Simmons. 1997. Elasticity and unfolding of single molecules of the giant muscle protein of titin. Nature 387: 308-312 |
36. | Wang, K.. 1996. Titin/connectin and nebulin: giant protein rulers of muscle structure and function. Adv. Biophys. 33: 123-132 |