Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106-4970
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ABSTRACT |
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Troponin T (TnT) is an essential element in the thin filament Ca2+-regulatory system controlling striated muscle contraction. Alternative RNA splicing generates developmental and muscle type-specific TnT isoforms differing in the hypervariable NH2-terminal region. Using avian fast skeletal muscle TnT containing a metal-binding segment, we have demonstrated a role of the NH2-terminal domain in modulating the conformation of TnT (Wang J and Jin JP. Biochemistry 37: 14519-14528, 1998). To further investigate the structure-function relationship of TnT, the present study constructed and characterized a recombinant protein in which the metal-binding peptide present in avian fast skeletal muscle TnT was fused to the NH2 terminus of mouse slow skeletal muscle TnT. Metal ion or monoclonal antibody binding to the NH2-terminal extension induced conformational changes in other domains of the model TnT molecule. This was shown by the altered affinity to a monoclonal antibody against the COOH-terminal region and a polyclonal antiserum recognizing multiple epitopes. Protein binding assays showed that metal binding to the NH2-terminal extension had effects on the interaction of TnT with troponin I, troponin C, and most significantly, tropomyosin. The data indicate that the NH2-terminal Tx [4-7 repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-His] extension confers a specific conformational modulation in the slow skeletal muscle TnT.
thin filament; epitope analysis; ELISA protein binding assay; metal affinity chromatography
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INTRODUCTION |
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CONFORMATIONAL MODULATION plays an important role in regulating protein function under physiological and pathological conditions. Troponin T (TnT), the tropomyosin (Tm) binding subunit of the troponin complex, is an essential element in the Ca2+ signal transduction pathway for the thin filament-based regulation of striated muscle contraction (20, 29, 39). Encoded by three (cardiac, slow, and fast skeletal muscle) genes and through developmentally regulated alternative mRNA splicing, a large number of TnT isoforms are expressed in vertebrate muscles (4, 6, 14, 19). Differential splicing generates a hypervariable NH2-terminal domain in the TnT polypeptide chain (4, 5, 18, 31, 34, 37). Inclusion or exclusion of an NH2-terminal acidic segment is responsible for a cardiac TnT isoform switch during both avian and mammalian heart development (6, 17). Similarly, a large-to-small, acidic-to-basic isoform transition of fast skeletal muscle TnT occurs during skeletal muscle development as the compound result of alternative splicing of multiple exons encoding the NH2-terminal variable region (37). Altered cardiac TnT isoform expression involving NH2-terminal alternative splicing also occurs during myocardial disorders (1, 30). The functional meaning of the TnT isoform regulation is not fully understood. The role of the alternatively spliced NH2-terminal variable region of TnT needs to be established for the physiological and pathological significance of different TnT isoforms.
By anchoring the troponin complex to the thin filament, TnT plays an organizer role in the Ca2+-regulatory system of muscle. Comprehensive studies have demonstrated that the COOH-terminal T2 fragment of TnT (see Fig. 3A) interacts with Tm, troponin I (TnI), troponin C (TnC), and actin (11, 15, 27, 28, 32). The central region of TnT (the CB2 fragment, see Fig. 3A) interacts with Tm (11, 27) and is required for the association of the troponin complex to Tm (7). In contrast, the NH2-terminal variable region of TnT (corresponding to the CB3 fragment, Fig. 3A) has not been found with any direct interaction with other thin filament proteins (27, 28). Deletion of this region does not abolish the basal activity of TnT in the Ca2+ activation of the thin filament (7) and the regulation of actomyosin ATPase (12, 26).
However, functional differences are present between TnT isoforms varying in the NH2-terminal structure. For example, ATPase activation was different in reconstituted thin filaments containing alternatively spliced TnT isoforms differing in their NH2-terminal primary structure (35). Alternative RNA splicing-generated TnT isoforms with NH2-terminal charge differences may alter the tolerance to acidosis (24). TnT isoforms with increased NH2-terminal-negative charge sensitize the myofibrillar contractile apparatus to Ca2+ (25). Using an avian fast skeletal muscle TnT containing a cluster of transition metal ion (Zn2+, Cu2+, Ni2+, and Co2+) binding sites [e.g., 4-7 repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-His, designated as Tx] in the NH2-terminal variable region (17), we have demonstrated that the metal-binding-induced structural changes within the NH2-terminal domain modulate the conformation of TnT (23, 38). In the present study, we further investigated this conformational modulation of TnT in a chimeric protein constructed with the chicken Tx metal-binding segment fused to the NH2 terminus of mouse slow skeletal muscle TnT (sTnT). The results demonstrate that the avian fast skeletal muscle TnT-originated Tx element, used as a representative NH2-terminal variable domain, confers conformational changes in mouse slow skeletal TnT.
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MATERIALS AND METHODS |
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Polyclonal and Epitope-Specific Monoclonal Anti-TnT Antibodies
A polyclonal anti-TnT antibody (RATnT) raised in rabbit against purified chicken breast muscle TnT was used in this study to monitor the conformation of multiple epitopes on TnT. This polyclonal antibody shows broad interactions with TnTs from different muscles across the vertebrate phyla (38).An anti-Tx monoclonal antibody (MAb) 6B8 (38) was used to identify the Tx segment at the NH2 terminus of the chimeric mouse slow skeletal muscle TnT and to examine the local conformational changes induced by the binding of Zn2+.
A cardiac and slow skeletal muscle TnT-specific MAb CT3 (18) was used to identify mouse sTnT and to monitor the conformational changes of a COOH-terminal epitope.
The isoform specificity of the three anti-TnT antibodies is summarized
in Fig. 1.
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The peptide specificity of MAb CT3 was determined by enzyme-linked
immunosorbent assay (ELISA). Bovine cardiac TnT was purified from adult
ventricular muscle as described previously (16) and digested with 5% CNBr in 70% formic acid. The CNBr-digested TnT fragments were fractionated by reverse phase HPLC on a C18
column. The four TnT fragments expected from cleavage at the Met
residues (Fig. 2A) were
recovered according to their mobility in a small-pore SDS-PAGE using
the Tris-Tricine buffering system (23) (Fig. 2B). The CNBr fragments were verified by amino acid analysis
showing residue ratios agreeing with that calculated from sequences
(data not shown). The CT3 MAb was partially purified from mouse
hybridoma ascites fluid by 50% ammonium sulfate precipitation and
labeled with horseradish peroxidase (HRP) (type VI, Sigma) as described by Avrameas and Ternynck (3). The resulting conjugate had
a molar ratio of HRP to immunoglobulin of 1.85, as calculated from its
optical absorbances at 405 nm and 280 nm. Since MAb CT3 recognizes cardiac and slow skeletal muscle TnTs across the vertebrate phyla (data
not shown), its epitope is expected to be in the conserved central or
COOH-terminal region. Therefore, the corresponding CNBr3 and CNBr4
fragments (Fig. 2A) were examined by ELISA for their
reactivity to CT3. The two purified TnT fragments were coated separately on microtiter plates for incubation with serial dilutions of
the HRP-labeled CT3 MAb. The following ELISA procedure was carried out
as described previously (16). The bound HRP-labeled CT3
MAb was quantified from triplicate experiments and plotted as titration
curves that show specific interaction of MAb CT3 with the COOH-terminal
CNBr4 fragment of TnT (Fig. 2C).
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Construction of a Prokaryotic Expression Vector Encoding sTnT with an NH2-Terminal Metal-Binding Extension
To construct a mammalian TnT model protein, a chicken Tx segment was fused to mouse sTnT (Tx-sTnT, Fig. 3). An expression plasmid was constructed using the T7 RNA polymerase-based pAED4 prokaryotic expression vector (23). As outlined in Fig. 4, a cDNA segment encoding the Tx metal-binding cluster containing four of the repeating His-Glu/Ala-Glu-Ala-His motifs was amplified from a chicken breast muscle TnT cDNA (TnT1, provided by Dr. L. B. Smillie, University of Alberta) by PCR using two synthetic oligo nucleotide primers (Tx-5F and Tx-R). This Tx-coding cDNA segment was cut with restriction enzyme Sph I at the 3' end and inserted into a pAED4 plasmid precut by restriction enzyme Nde I, blunted with mung bean exonuclease, and then cut with Sph I. DNA sequencing confirmed the proper insertion of the Tx-coding sequence into the expression vector (Fig. 4). This modified expression vector was then cleaved by restriction enzymes Sph I and Sma I and a short synthetic Sph I-Nde I linker was inserted. A cDNA encoding mouse sTnT (18) was isolated as an Nde I-EcoR I fragment and inserted into the Nde I-EcoR I cut expression vector (Fig. 4). The final construct was verified by DNA sequencing.
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Expression of Tx-sTnT in Escherichia coli and Metal Affinity Purification
As described previously (23), a large culture of E. coli BL21(DE3)pLysS freshly transformed with the pAED4 Tx-sTnT expression plasmid was induced with isopropyl-1-thio-Bacterial Expression and Purification of Mouse Slow Skeletal Muscle TnT
BL21(DE3)pLysS E. coli was transformed with a recombinant pAED4 expression vector encoding mouse sTnT (18) and cultured as above. The IPTG-induced bacteria were collected and washed with cold ethanol and acetone. The dried acetone powder was extracted by 6 M urea, 0.1 mM EDTA, 15 mMPreparation of Other TnT Isoforms, Tm, TnI, and TnC
Tx-positive TnT1e17 and Tx-negative TnT4e17. The bacterial expression and purification of cloned chicken TnT1e17 and TnT4e17 were carried out as described previously (38).
Rabbit fast skeletal muscle TnT. TnT was purified from the back muscle of the New Zealand White rabbit as described by Pearlstone and Smillie (27).
Chicken -Tm.
Adult chicken heart tissue (ventricles) was used to purify
-Tm by
heat-denaturation enrichment and ion-exchange chromatography as
described by Smillie (33).
Chicken skeletal muscle TnI. Adult chicken breast muscle was used for TnI purification by the ion-exchange chromatography method described in Mak et al. (22).
Chicken fast skeletal muscle TnC. A recombinant pET3 expression plasmid encoding chicken fast skeletal muscle TnC (provided by Dr. L. B. Smillie, University of Alberta) was used to express TnC in E. coli. The bacterial culture and induction were similar to the TnT expression. The bacterial cells harvested were lysed in 6 M urea, 30 mM Tris · HCl, pH 8.0, and 2 mM MgCl2 by three passes through a French press. The clarified lysate was loaded onto a DE52 ion-exchange column equilibrated in the same buffer and eluted by a 0-300 mM KCl gradient. The fractions containing the TnC peak were identified by SDS-PAGE, collected, and dialyzed against double distilled water. The sample was then concentrated by lyophilization for further fractionation on a gel filtration column (Sephadex G75, Pharmacia-Amersham) in 0.5 M KCl, 20 mM Tris · HCl, pH 8.0, and 2 mM MgCl2. The purified TnC was dialyzed and lyophilized as above.
Western Blotting
Total protein extracts from muscle tissue or E. coli were run on Laemmli SDS-PAGE. The resolved protein bands were transferred onto nitrocellulose membrane. The blocking, incubation with anti-TnT antibodies and alkaline phosphatase-labeled second antibodies (Sigma), high-stringency washes, and the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate reactions were performed as described previously (24, 38).ELISA Epitope Analysis to Characterize the Metal-Induced Conformational Changes of TnT
An ELISA-based epitope analysis (38) was carried out to monitor conformational changes in the Tx-sTnT-induced by the binding of metal ions to the NH2-terminal Tx extension. The original conformation change within the Tx metal-binding cluster and the secondary conformational changes induced in other domains of Tx-TnT were analyzed using the anti-Tx MAb 6B8 and MAb CT3 against an epitope on sTnT distant from the NH2-terminal Tx extension or a polyclonal antibody RATnT, respectively. Similar to a protocol described previously (38), purified Tx-sTnT in buffer A (0.1 M KCl, 3 mM MgCl2, and 10 mM PIPES, pH 7.0) was coated on microtiter plates in the presence or absence of ZnCl2. After washing, the remaining plastic surface was blocked with 1% BSA and 0.05% Tween 20 in buffer A. Subsequently, the immobilized Tx-sTnT was incubated with serial dilutions of antibody 6B8, CT3, or RATnT. Following washes with buffer A plus 0.05% Tween 20 (buffer T) to remove any free first antibody, the plates were further incubated with HRP-conjugated anti-mouse or anti-rabbit immunoglobulin second antibody. The free second antibody was washed away with buffer T and followed by H2O2-2,2'-azinobis-(3-ethylbenzthiazolinesulfonic acid) (ABTS) substrate reaction. All of the experiments were done in triplicate. Absorbance at 405 nm (A405nm) for each assay well was recorded at a series of time points by an automated microplate reader (Bio-Rad Benchmark). A405nm values in the linear range of the color development were used to plot the antibody affinity titration curves to quantify the interaction between the antibodies and the specific epitopes on the Tx-sTnT molecule.Wild-type mouse sTnT, chicken fast skeletal muscle TnT isoforms TnT1e17 and TnT4e17, as well as rabbit fast skeletal muscle TnT were analyzed in parallel as controls. The two chicken fast skeletal muscle TnTs are identical except for the inclusion (TnT1e17) or exclusion (TnT4e17) of the NH2-terminal Tx segment.
Dual-Antibody ELISA to Analyze the Conformational Change of Tx-sTnT Induced by Binding of MAb 6B8 to the NH2-Terminal Tx Extension
Using the specific anti-Tx and anti-TnT antibodies raised in different species, we were able to detect changes in the conformation of mouse sTnT resulting from a structural reconfiguration of the NH2-terminal Tx extension by the binding of MAb 6B8. In the dual-antibody ELISA (38), the Tx-sTnT was coated on microtiter plates, and the wells were blocked as above. The coated Tx-sTnT was first incubated with serial dilutions of the mouse MAb 6B8 in buffer T containing 0.1% BSA followed by the addition of the RATnT rabbit polyclonal antiserum at a predetermined dilution. After a further incubation, two parallel sets of assay wells were washed with buffer T and incubated with HRP-conjugated anti-rabbit or anti-mouse immunoglobulin second antibody (Sigma), respectively, to selectively detect the bound RATnT or 6B8 antibodies. After final washes with buffer T, the plates were developed with H2O2-ABTS substrates, and A405nm was measured as above. The relationship between the binding of MAb 6B8 to the NH2 terminus and the reactivity of Tx-sTnT to the RATnT polyclonal antiserum was plotted.Direct ELISA experiments were carried out in parallel to analyze the effect of the 6B8 MAb-Tx-sTnT interaction on the conformation of the COOH-terminal CT3 MAb epitope of Tx-sTnT. The microtiter plates were coated with Tx-sTnT, blocked with BSA, and incubated with serial dilutions of MAb 6B8 as above. A pretitrated dilution of the HRP-labeled CT3 MAb was then added alongside the 6B8 MAb. After washing with buffer T, H2O2-ABTS substrate reaction and A405nm measurement were performed as above.
ELISA-Mediated Solid-Phase Protein Binding Assays
ELISA-based solid-phase protein binding assay (23, 24, 38) was used to characterize the effect of the NH2 terminus-based conformational changes on the interaction of TnT with TnI, TnC, and Tm. Microtiter plates were coated with Tx-sTnT in buffer A. After washes to remove the excess Tx-sTnT and blocking with buffer T containing 1% BSA, the plates were incubated with serial dilutions of chicken fast skeletal muscle TnI, fast skeletal muscle TnC, or ![]() |
RESULTS |
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The Tx-sTnT Chimeric Protein
The Tx-sTnT fusion protein was constructed and expressed in E. coli to test the hypothesis that structural modifications in the NH2-terminal domain of TnT can modulate the conformation of TnT. Although the expression level was relatively low, Western blots using MAb CT3 detected the recombinant protein in the bacterial lysate. The apparent molecular mass of the Tx-sTnT protein was higher than the wild-type mouse sTnT high-Mr isoform 1, as expected (Fig. 5A).
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Despite the low level expression, the Zn2+ affinity
chromatography achieved an effective recovery and one-step purification of the Tx-sTnT protein from the E. coli lysate (Fig.
5B). The results demonstrated that the Tx segment attached
to the NH2 terminus of sTnT retains its highly selective
strong binding to the immobilized Zn2+ ions, the same as
when it resides in the chicken breast muscle TnT (17).
Since the Tx metal-binding cluster contains repeating His pairs
separated by three amino acids in an -helix (Fig. 3A), it
confers an ideal molecular structure to bind transition metal ions
(2, 13). The metal-binding property of Tx-sTnT further demonstrated that the Tx segment acts as an independent, modular structure in a fusion protein. Results of the metal affinity
chromatography in Fig. 5B showed that the interaction of Tx
with immobilized Zn2+ ion is significantly stronger (eluted
at 40-50 mM imidazole) than that of the single His pair (eluted at
~10 mM imidazole; Ref. 2). Coupled with the significantly higher
binding affinity of Tx to Ni2+ (17) compared
with the widely used His-(6-8) tag, the Tx tag is
highly effective for metal affinity isolation of recombinant proteins
and peptides.
The construction of the Tx-sTnT was verified by its apparent molecular weight in SDS-PAGE as well as its reactivity to anti-TnT and anti-Tx antibodies in Western blotting (Fig. 5C). The Tx-sTnT protein retained activity to bind Tm, TnC, and TnI as shown by the protein binding assays (see below), further proving the accurate engineering of the Tx-sTnT protein.
NH2-Terminal Conformational Change Induced by the Binding of Metal Ion to Tx and Its Secondary Effects on the Conformation of TnT
We have previously shown that binding of Zn2+ to the Tx element of chicken breast muscle TnT induced a local conformational change within the Tx structure (23, 38). This local allosteric change is preserved when the Tx segment is fused to the NH2 terminus of mouse sTnT, This is demonstrated by the metal-induced decrease of the affinity of Tx-sTnT to the anti-Tx MAb 6B8. Figure 6A shows that when the Tx-sTnT was incubated with 0.1 mM ZnCl2, ELISA epitope analysis detected a dramatically decreased binding of MAb 6B8 compared with that in the control incubated with 0.1 mM EGTA. This result demonstrates that the Tx structure retains its conformational feature when isolated from the natural Tx-positive avian fast skeletal muscle TnT.
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The results in Fig. 6B demonstrate a remote conformational modulation of the Tx-sTnT by the binding of Zn2+ to the NH2-terminal Tx segment. Compared with the EGTA-treated sample, the Zn2+-treated Tx-sTnT showed significantly decreased affinity to MAb CT3 against an epitope in the COOH-terminal CNBr4 fragment of TnT (Fig. 2). The identical reactivity of CT3 to the three cloned mouse sTnT isoforms (Fig. 5C) which vary in their NH2-terminal structure (18) further suggests that the CT3 epitope is not directly related to the NH2-terminal variable region when it resides in an intact TnT molecule. Therefore, the conformation of the distant CT3 epitope was modulated as a secondary effect of the conformational change in the NH2-terminal Tx extension. This result clearly indicates that Zn2+-induced reconfiguration of the NH2-terminal Tx extension confers a conformational modulation of other domains of the Tx-sTnT protein.
The results in Fig. 6C further show that the binding of Zn2+ to the NH2-terminal Tx extension in Tx-sTnT altered the binding affinity of the RATnT polyclonal antibody, implying conformational changes in multiple epitopes along the TnT molecule. Together with the CT3 MAb data, the results indicate that the molecular conformation of Tx-sTnT was extensively changed as a consequence of the local structure change in the NH2-terminal Tx extension.
Similar epitope conformational changes induced by the binding of metal ion to Tx were seen for the Tx-positive fast skeletal muscle TnT1e17 (Fig. 6D). In contrast, the presence of Zn2+ did not result in significant change in the affinity of the RATnT antibody to the Tx-negative TnT4e17 (Fig. 6E). This is consistent with our previous demonstration that the reactivity of other Tx-negative chicken fast skeletal muscle TnTs to RATnT was not affected by the presence or absence of Zn2+ (23, 38). The incubation with ZnCl2 did not cause a significant change in the binding affinity of control, wild-type mouse sTnT to MAb CT3 or the polyclonal antiserum RATnT (Fig. 6, F and G, respectively). Similarly, mammalian (rabbit) fast skeletal muscle TnT (which does not contain the Tx metal-binding element) showed no response to Zn2+, demonstrated by its unchanged affinity to T12 MAb (Fig. 6H). These results preclude any effect by nonspecific interaction between Zn2+ and TnT.
Effect of the Binding of Anti-Tx MAb on the Conformation of Tx-sTnT
Binding of the anti-Tx MAb 6B8 to Tx-sTnT resulted in significant changes in the binding affinity of the polyclonal RATnT antibody. The ELISA binding curves (Fig. 7) demonstrate that with increasing binding of 6B8 to the Tx extension on Tx-sTnT, the reactivity of RATnT becomes weaker. To exclude the competition effect between 6B8 MAb and RATnT for the Tx epitope, we also observed this pattern by direct ELISA experiments analyzing the effect of 6B8 binding on the affinity of MAb CT3 for Tx-sTnT (Fig. 7). Although the Tx-metal interaction is not a universal mechanism in regulating TnT structure and function, the similar secondary effects induced by the binding of Zn2+ or a protein (immunoglobulin) with the NH2-terminal Tx extension suggest that structural modification of the NH2-terminal region can modulate the molecular conformation of TnT.
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Effect of Zn2+ Binding on the Interaction of Tx-sTnT to TnI, TnC, and Tm
The ELISA protein binding experiments showed that treatment of Tx-sTnT with ZnCl2 altered the binding affinity to TnI, TnC, and Tm (Fig. 8, A, B, and C, respectively). The changes in TnI- and Tm-binding affinity induced by Zn2+ binding to the NH2-terminal Tx are similar to those obtained from studies on chicken breast muscle TnT (23, 38). The Zn2+-induced change in the binding affinity of Tx-sTnT for TnC in the absence of Ca2+ demonstrates that the NH2-terminal structure-based modulation of TnT molecular conformation also affects the interaction with TnC. This finding is in agreement with the central organizer role of TnT in the thin filament regulatory system (36). Based on the data shown in Fig. 6, B and C, the decreases in TnI-, TnC-, and Tm-binding affinity are due to secondary conformational changes in multiple functional domains of TnT. The protein binding data demonstrate that local structural changes within the NH2-terminal Tx extension can modulate not only the molecular conformation but also the interaction of TnT within the thin filament regulatory system. Compared with the significant change in the TnT-Tm interaction (as measured by the concentration required for achieving 50% maximum binding during equilibrium incubation and the final amount of solid-phase binding after the nonequilibrium washes), the effects on TnI- and TnC-binding affinity were not as dramatic. The more effective modulation of the TnT-Tm interaction suggest that the Tm-binding site in the central domain of TnT is highly responsive to the NH2-terminal structural changes and may be a major target of the TnT isoform regulation. This hypothesis is consistent with the model that TnT is an extended molecule (8) and the fact that the Tm-binding site in the central domain of TnT is required for the strong interaction between TnT and Tm (7, 11, 12, 27).
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DISCUSSION |
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This study demonstrates that Tx-sTnT mimics biochemical properties of the Tx-positive avian fast skeletal muscle TnT as detected by the antibody probes, and the interaction with TnC, TnI, and Tm is also altered. The effects on the regulation of actomyosin ATPase and muscle contractility remains to be investigated to correlate to the functional difference of TnT isoforms with NH2-terminal structure variation.
The TnI, TnC, and Tm binding assays showed that the Tx-sTnT with an NH2-terminal peptide extension retained interactions with other thin filament regulatory proteins. This adds evidence for the structural tolerance of the NH2-terminal hypervariable region of TnT (7, 9, 12, 17). This feature allows the evolution of a large number of alternatively spliced variants in different muscle fibers and at different developmental stages. The TnT isoforms with different NH2-terminal structures would therefore contribute to evolutionary adaptations to enrich the contractile feature of different muscle fibers. Accordingly, the Tx-sTnT construct may be tested as a model protein for the structure-function relationship of TnT isoforms.
Although the antigen-antibody interaction may be affected by many environmental factors, the spatial fit between the antigenic epitope and the antibody paratope is an essential requirement. Therefore, antibody epitope analysis provides a useful tool for investigating protein conformational changes (10). This is especially valuable before high-resolution crystallography and nuclear magnetic resonance structural information is available for TnT conformation and the structure-function relationship of the NH2-terminal variable region.
When the Tx segment is placed as an extension of the NH2 terminus of a full-length mammalian sTnT, the binding of Zn2+ retained the ability to reconfigure the local three-dimensional structure (Fig. 6A). This primary conformational change also retained the ability to induce secondary effects on the conformation of other domains of TnT, as demonstrated by the change in binding affinity of MAb CT3 (Fig. 6B) against an epitope in the COOH-terminal region and the polyclonal antibody RATnT against multiple epitopes (Fig. 6C). This modulation of TnT conformation indicates that the allosteric structural changes within the NH2-terminal Tx extension can propagate into the sTnT structure, even when the Tx segment is placed as an attachment to the natural NH2 terminus of the mouse sTnT.
When the binding of anti-Tx MAb 6B8 was used as an alternative method to confer a reconfiguration of the NH2-terminal structure of Tx-sTnT, the conformation of Tx-sTnT was also significantly altered (Fig. 7). This result is similar to that obtained with the chicken breast muscle TnT (38) in which the Tx segment resides in the middle of the NH2-terminal variable region. The data strongly support the hypothesis that it is the change of the NH2-terminal domain structure, rather than an effect restricted to the metal ion binding, that modulates the conformation of the TnT molecule.
As demonstrated by the specific epitope analyses, the decreased but still saturable binding to the Zn2+-charged Tx-sTnT (Fig. 6, A and B) suggests that two allosteric conformational states may exist for both NH2- and COOH-terminal domains of TnT. The significance of the conformational states of TnT was further shown by the TnT-TnI and TnT-Tm interactions (Fig. 8, A and C, respectively) which demonstrate two saturable states of binding, in the presence or absence of Zn2+, respectively. This two-conformational states model is also consistent with the observation that the responses of TnT to serial decreases of environmental pH showed quantum changes (24).
In summary, the NH2-terminal-based TnT conformational modulation model supports the hypothesis that the alternatively spliced NH2-terminal hypervariable region of TnT has evolved as a regulatory structure to diversify the contractile features of muscle. Although this region can tolerate various structural changes and its removal does not abolish the core function of TnT (9, 26), it plays an active role by modulating TnT molecular conformation through providing NH2-terminal structural variants.
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ACKNOWLEDGEMENTS |
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We thank Dr. Larry Smillie for the TnT1 cDNA and the TnC expression plasmid, Dr. Jim Lin for the CH1 and T12 MAbs, and Dr. Natalie Strynadka for the modeling of Tx metal-binding structure shown in Fig. 3A.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J.-P. Jin, Dept. of Physiology and Biophysics, Case Western Reserve Univ. School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970 (E-mail: jxj12{at}po.cwru.edu).
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.
Received 6 January 2000; accepted in final form 14 April 2000.
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