Wen-Ji Dong

Conformation of the Regulatory Domain of Cardiac Muscle Troponin C in Its Complex with Cardiac Troponin I

Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca2+-dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca2+. However, in the presence of bound cardiac TnI (cTnI), activator Ca2+induces significant increases in the distances and a substantial opening of the N-domain. This open conformation within the cTnC·cTnI complex has properties favorable for the Ca2+-induced interaction with an additional segment of cTnI. Thus, the binding of cTnI to cTnC is a prerequisite to achieve a Ca2+-induced open N-domain similar to that previously observed in fsTnC with no bound fsTnI. This role of cardiac TnI has not been previously recognized. Our results also indicate that structural information derived from a single protein may not be sufficient for inference of a structure/function relationship.

Observation of a Partially Opened Triple-helix Conformation in 1→3-β-Glucan by Fluorescence Resonance Energy Transfer Spectroscopy

This study used fluorescence resonance energy transfer (FRET) spectroscopy as an indirect method to investigate the effect of NaOH treatment on the conformation of a triple-helix (1→3)-β-d-glucan and then evaluated the effect of conformation on biological activity. Previous studies have suggested that treatment of the triple-helix glucans with NaOH produces single-helix conformers. FRET spectra of the triple-helix glucan, laminarin, doubly labeled with 1-aminopyrene as donor probe and fluorescein-5-isothiocyanate as acceptor probe attached at the reducing end, showed that a partially opened triple-helix conformer was formed on treatment with NaOH. Increasing degrees of strand opening was associated with increasing concentrations of NaOH. Based on these observations we propose that a partially opened triple-helix rather than a single helix, is formed by treating the triple-helix glucans with NaOH. After neutralizing the NaOH, changes in FRET indicated that the partially opened conformer gradually reverts to the triple-helix over 8 days. Laminarian was stabilized at different degrees of partial opening and its biological activity examined using theLimulus amebocyte lysate assay and nitric oxide production by alveolar macrophage. Both Limulus amebocyte lysate activity and nitric oxide production were related to the degree of opening of the triple-helix. Partially open conformers were more biologically active than the intact triple-helix.

De novo design of peptides targeted to the EF hands of calmodulin.

This report describes the use of the concept of inversion of hydropathy patterns to the de novo design of peptides targeted to a predetermined site on a protein. Eight- and 12-residue peptides were constructed with the EF hands or Ca2+-coordinating sites of calmodulin as their anticipated points of interaction. These peptides, but not unrelated peptides nor those with the same amino acid composition but a scrambled sequence, interacted with the two carboxyl-terminal Ca2+-binding sites of calmodulin as well as the EF hands of troponin C. The interactions resulted in a conformational change whereby the 8-mer peptide-calmodulin complex could activate phosphodiesterase in the absence of Ca2+. In contrast, the 12-mer peptide-calmodulin complex did not activate phosphodiesterase but rather inhibited activation by Ca2+. This inhibition could be overcome by high levels of Ca2+. Thus, it would appear that the aforementioned concept can be used to make peptide agonists and antagonists that are targeted to predetermined sites on proteins such as calmodulin.

A kinetic model for the binding of Ca2+ to the regulatory site of troponin from cardiac muscle.

The kinetics of the binding of Ca2+ to the single regulatory site of cardiac muscle troponin was investigated by using troponin reconstituted from the three subunits, using a monocysteine mutant of troponin C (cTnC) labeled with the fluorescent probe 2-[(4′-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-35. The kinetic tracings of binding experiments for troponin determined at free [Ca2+] > 1 μm were resolved into two phases. The rate of the fast phase increased with increasing [Ca2+], reaching a maximum of about 35 s−1 at 4 °C, and the rate of the slow phase was approximately 5 s−1 and did not depend on [Ca2+]. Dissociation of bound Ca2+ occurred in two phases, with rates of about 23 and 4 s−1. The binding and dissociation results obtained with the binary complex formed between cardiac troponin I and the IAANS-labeled cTnC mutant were very similar to those obtained from reconstituted troponin. The kinetic data are consistent with a three-step sequential model similar to the previously reported mechanism for the binding of Ca2+ to a cTnC mutant labeled with the same probe at Cys-84 (Dong et al. (1996) J. Biol. Chem. 271, 688–694). In this model, the initial binding in the bimolecular step to form the Ca2+-troponin complex is assumed to be a rapid equilibrium, followed by two sequential first-order transitions. The apparent bimolecular rate constant is 5.1 × 107 m −1 s−1, a factor of 3 smaller than that for cTnC. The rates of the first-order transitions are an order of magnitude smaller for troponin than for cTnC. These kinetic differences form a basis for the enhanced Ca2+ affinity of troponin relative to the Ca2+ affinity of isolated cTnC. Phosphorylation of the monocysteine mutant of troponin I by protein kinase A resulted in a 3-fold decrease in the bimolecular rate constant but a 2-fold increase in the two observed Ca2+ dissociation rates. These changes in the kinetic parameters are responsible for a 5-fold reduction in Ca2+ affinity of phosphorylated troponin for the specific site.

Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C

The regulatory domain of troponin C (TnC) from chicken skeletal muscle was studied using genetically generated mutants which contained a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The large quantum yield of the 2-Mg state is due to a relatively small nonradiative decay rate and consistent with the emission peak at 331 nm. The intensity decay of this state is monoexponential with a single lifetime of 5.65 ns, independent of wavelength. In the 4-Ca state, the decay is biexponential with the mean of the two lifetimes increasing from 4.54 to 4.92 ns across the emission band. The decay-associated spectrum of the short lifetime is red-shifted by 19 nm relative to the steady-state spectrum. The decay of Trp-52 is biexponential in the 2-Mg state and triexponential in the 4-Ca state. The decay of Trp-90 requires three exponential terms for a satisfactory fit, but can be fitted with two exponential terms in the 4-Ca state. The lower quantum yields (< 0.15) of these two tryptophans are due to a combination of smaller radiative and larger nonradiative decay rates. The results from Trp-22 suggest a homogeneous ground-state indole ring in the absence of bound Ca2+ at the regulatory sites and a ground-state heterogeneity induced by activator Ca2+. The Ca(2+)-induced environmental changes of Trp-52 and Trp-90 deviate from those predicted by a modeled structure of the 4-Ca state. The anisotropy decays of all three tryptophans show two rotational correlation times. The long correlation times (phi 1 = 8.1-8.3 ns) derived from Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. TnC becomes more asymmetric upon binding activator Ca2+ (phi 1 = 10.1-11.6 ns). The values of phi 1 obtained from Trp-52 are 3-4 ns shorter than those from Trp-22 and Trp-90, and these reduced correlation times may be related to the mobility of the residue and/or local segmental flexibility.

The Calcium-Saturated cTnI/cTnC Complex: Structure of the Inhibitory Region of cTnI

The contiguous inhibitory and regulatory regions of troponin I in the heterotrimeric troponin complex play a critical role in Ca(2+) activation of striated muscle. Knowledge of the structure of this critical region within the complex will enhance efforts toward understanding regulatory mechanisms. Toward this goal, we have used simulated annealing to study the structure of the inhibitory and regulatory regions of cardiac muscle troponin I in the calcium-saturated complex formed between cardiac troponin C and cardiac troponin I. We have incorporated distances determined experimentally by Förster resonance energy transfer in the full-length complex, rather than using peptides derived from cTnI. For these models, we assume a helix-loop-helix conformation for the inhibitory region. We have found several structures that satisfy the experimental constraints fairly well. Although it is not possible to eliminate any of these models at this time, future studies with additional experimental restraints will yield insights on the mechanisms of calcium regulation in cardiac muscle.

Fret Studies of Conformational Transitions in Proteins

In recent years, fluorescence spectroscopy has been increasingly recognized as a useful tool for probing changes in localized and global conformations of macromolecules. Both intrinsic and extrinsic fluorophores can be used for these studies and for determination of intermolecular interactions. In proteins, the three aromatic amino acid residues have emission properties that can be exploited to reveal localized conformational changes. Such changes are usually described by changes in some basic spectroscopic properties of the fluorophores, rather than by a quantitative parameter to indicate the magnitude of the changes. When two fluorophores attached to the same protein or two proteins in a complex are used, the separation between their sites (R) can be quantified from measurements of the Forster type of resonance energy transfer (FRET) from an initially excited donor fluorophore (D) to an acceptor fluorophore (A). This transfer occurs without the appearance of a photon and results from a long-range dipole-dipole interaction between the donor emission dipole and the acceptor absorption dipole. Because the acceptor emission dipole is not involved in the transfer, the acceptor probe need not be fluorescent if its absorption spectrum overlaps with the donor emission spectrum. The rate of transfer (and the efficiency of transfer) is dependent upon the extent of the spectral overlap between the long-wavelength region of donor emission spectrum and the short-wavelength region of the acceptor absorption spectrum.