Monoclonal Antibodies as Probes to Study Ligand-Induced Conformations of Troponin Subunits

Figures

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  Figure 1. Antibody as probe to detect protein conformational change. Antibody–antigen binding is a protein–protein interaction whose affinity is determined by 3-D structural fitting of the antigenic epitope and the variable region of antibody. Changes in the epitope conformation can be reflected in the antibody binding affinity, where an alteration of the epitope conformation can shift an antibody–antigen interaction from a better fit with higher affinity (left) to a partial fit with lower affinity (right) and vice versa.

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  Figure 2. ELISA-based mAb epitope analysis. Protein of interest was coated on a microtiter plate in a buffer containing the desired additives, which can be used as the buffer throughout the entire assay or modified in later steps. After blocking the plate, the mAb conformational probe was incubated in serial dilutions, followed by washes and incubation with horse radish peroxidase (HRP)-conjugated secondary antibody. After final washes, the binding of mAb probes was detected via H2O2-ABTS colorimetric substrate reaction and recording the development of absorbance at 420 nm.

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  Figure 3. Localized surface plasmon resonance-based mAb epitope analysis. Protein of interest was attached to a carboxy-functionalized gold nanoparticle via covalent bonding with lysine residues. The protein was permitted to equilibrate in running buffer, and then the mAb probe diluted in the same buffer was permitted to flow over the chip and interacts with the protein. Interactions were measured via local changes in refractive index that are reflected in optical signal detection. Running buffer was then flushed over the chip to promote mAb dissociation. The resulting signal measured by the optical detector is plotted against time to calculate antibody association and dissociation rate constants.

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  Figure 4. Structure of chicken (Gallus gallus) fast skeletal muscle TnC. (A) A linear map showing locations of epitopes recognized by anti-TnC mAbs 4E7, 2C3, and 2D10 used as conformational probes in the present study. Fast TnC consists of N and C terminal domains containing four E-F hand Ca2+-binding motifs, along with a central helical linker to connect the domains. N-terminal (amino acids 1–91) and C-terminal (89–163) fragments were employed to map epitopes of the mAbs. (B) A crystal structure of fast TnC in the Ca2+-saturated state (PDB: 1YTZ; Ca2+ ions are present as green spheres). The N and C termini, and the boundary of the N- and C-terminal fragments used for mAb epitope mapping are indicated.

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  Figure 5. Conformational effect of Ca2+ and/or Mg2+ binding to fast TnC detected in microtiter plate ELISA. (A) The ELISA titration curves show that mAb 4E7 against the N domain of fast TnC exhibits a low affinity in the metal-free condition. The presence of Mg2+, which binds to the C domain sites, significantly increased the affinity of mAb 4E7, indicating an effect on the N-domain conformation. Binding of Ca2+ to the N-domain sites in the presence of 3 mM Mg2+ further increased the affinity of mAb 4E7. The highest affinity of mAb 4E7 was seen in the four-Ca2+ state, indicating that the occupations of the C domain sites by Ca2+ or Mg2+ have different conformational effect on fast TnC. (B) mAb 2D10 against the C domain of fast TnC shows higher affinities in the four-Ca2+ or Ca2+ + Mg2+ state than that of Mg2+ alone while the metal free-apo state has the lowest affinity, indicating both ions bind to the C-domain with similar conformational effect whereas Ca2+ binding to the N domain increases the affinity for mAb 2D10. (C) mAb 2C3 detected a conformation-independent epitope that displays similar affinity for fast TnC in the presence or absence of Ca2+ and Mg2+ ions.

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  Figure 6. Conformational effect of Ca2+ binding to fast TnC detected in quantitative LSPR assay. (A) Changes in buffer pCa did not affect the binding of mAb 2C3, consistent with the observation in ELISA that mAb 2C3 binding to fast TnC remains constant regardless of ion binding. In addition, the binding signal of mAb 2C3 confirms constant protein coating on the LSPR chip. (B) In contrast, the binding of mAb 4E7 against the N domain of fast TnC changes with the change in pCa. A significant decrease in association rate and increase in dissociation rate at pCa 9.0 vs. pCa 4.0 are detected, indicating weaker binding of the mAb probe in the Ca2+-free state. (C) The physiological range pCa titration curve of mAb 4E7 binding with LSPR indicates that fast TnC changes conformation when [Ca2+] increases. The maximum binding of mAb 4E7 to the fast TnC chip, Bmax, increased in a gradual manner to a plateau level when [Ca2+] reached pCa 7.0, while KD, the equilibrium dissociation constant, started to decrease when [Ca2+] increases to pCa 8.0 and rapidly minimized at pCa 7.0. (D) The ELISA titration curves of mAb 4E7 binding to fast TnC at the same range of pCa (left panel) show a dramatic increase in…

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  Figure 7. Linear structure of chicken fast skeletal muscle TnT8e16 and epitope mapping of anti-TnT mAbs. (A) Locations of the epitopes recognized by anti-TnT mAbs used in the present study are indicated on a linear map of chicken fast TnT8e16. The Tx segment of chicken fast TnT, consisting of seven repeated H(E/A)EAH transition metal binding motifs, is encoded by alternatively-spliced exons. (B) Western blotting and (C) ELISA using intact chicken TnT8e16 and N-terminal T1 fragment N165 demonstrated that mAbs 2C8, 4C3, and 3C11 epitopes are contained within the N165 peptide, while the 3H12 epitope is outside of N165. mAb 3E4 had no reaction with N165 due to the lack of exon 7-encoded segment, serving as a background control.

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  Figure 8. Conformational effect of metal ion binding to chicken fast skeletal muscle TnT8e16 detected by ELISA epitope analysis. (A) Zn2+-binding to the N-terminal Tx segment of TnT8e16 resulted in a dramatic decrease in the affinity of anti-Tx mAb 3C11 as compared to the ethylenediaminetetraacetic acid (EDTA) control. (B) mAb 3E4 raised against an epitope related the exon 7-encoded segment downstream of the Tx metal binding site also showed decreased affinity with the binding of Zn2+, though less dramatic, reflecting conformational effects propagated from the metal-Tx binding. (C) Zn2+ binding to the Tx segment enhanced the binding of mAb 2C8 against the exons 10–11 encoded segment further downstream in comparison to the EDTA control. (D) mAb 4C3 shows an effect similar to that of mAb 2C8. (E) mAb 3H12 against an epitope in the T2 domain of TnT8e16 remote from the Tx segment did not detect any Zn2+ binding-induced conformational change. In addition to reflecting that the T2 domain of TnT may conform largely independent of the N-terminal variable region, the result served as a protein coating control.