Construction and characterization of a spectral probe mutant of troponin C: application to analyses of mutants with increased calcium affinity

Abstract

A spectral probe mutant (F29W) of chicken skeletal muscle troponin C (TnC) has been prepared in which Phe-29 has been substituted by Trp. Residue 29 is at the COOH-terminal end of the A helix immediately adjacent to the Ca2+ binding loop of site I (residues 30-41) of the regulatory N domain. Since this protein is naturally devoid of Tyr and Trp, spectral features can be assigned unambiguously to the single Trp. The fluorescent quantum yield at 336 nm is increased almost 3-fold in going from the Ca(2+)-free state to the 4Ca2+ state with no change in the wavelength of maximum emission. Comparisons of the Ca2+ titration curves of the change in far-UV CD and fluorescence emission indicated that the latter was associated only with the binding of 2Ca2+ to the regulatory sites I and II. No change in fluorescence was detected by titration with Mg2+. The Ca(2+)-induced transitions of both the N and C domains were highly cooperative. Addition of Ca2+ also produced a red shift in the UV absorbance spectrum and a reduction in positive ellipticity as monitored by near-UV CD measurements. The fluorescent properties of F29W were applied to an investigation of five double mutants: F29W/V45T, F29W/M46Q, F29W/M48A, F29W/L49T, and F29W/M82Q. Ca2+ titration of their fluorescent emissions indicated in each case an increased Ca2+ affinity of their N domains. The magnitude of these changes and the decreased cooperativity observed between Ca2+ binding sites I and II for some of the mutants are discussed in terms of the environment of the mutated residues in the 2Ca2+ and modeled 4Ca2+ states.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II

“…The titration curves of IFcTnC and ScTnC are superimposable, and the K 1 ⁄2 values of these curves are similar, demonstrating that the differences in sequence between IFcTnC and ScTnC do not appear to affect Ca 2ϩ affinity. IFcTnC also contains Asn 2 , Gln 29 , and Asp 30 , suggesting that one or a combination of these amino acids is responsible for the high Ca 2ϩ affinity of ScTnC and IFcTnC.…”

“…The replacement of Leu 29 and Gly 30 in McNTnC with Gln and Asp, respectively, decreased the K 1 ⁄2 by 0.09 pCa units ( Table 1). The Ca 2ϩ titration curve of the mutant protein, L29Q/G30D McNTnC, was shifted to the right of the Ca 2ϩ titration curves for both McNTnC and ScNTnC (Fig.…”

“…The differences in Ca 2ϩ affinity between mammals and fish can be attributed to amino acid substitutions in the NH 2 -terminal domain, because the removal of the COOH terminus (amino acids 90-161) through the creation of cNTnC mutants maintained the difference in Ca 2ϩ affinity. Gln 29 and Asp 30 were also demonstrated as being required for the higher Ca 2ϩ affinity of ScNTnC.…”

Calcium regulation of troponin and its role in the dynamics of contraction and relaxation

“…Studies on cTnC mutant forms show that by decreasing the Ca 2ϩ dissociation rate without changing the Ca 2ϩ association rate, TnC exhibits an increased Ca 2ϩ affinity (4,10,24). From these kinds of studies, Luo et al (18) provided direct evidences that decreasing the off-rate by site-directed mutagenesis can slow down the relaxation kinetics in skinned fibers, while increasing the off-rate of TnC does not affect relaxation kinetics, probably, because any accelerating effect on relaxation would be damped by the rate limit of cross-bridge detachment kinetics.…”

Cloning and expression of chicken skeletal muscle troponin I in Escherichia coli: The role of rare codons on the expression level

“…The fact that the three troponin subunits can refold and reassociate in vitro (Greaser & Gergely, 1971) has allowed the use of troponin subunits produced in Escherichia coli to study the molecular mechanism of this regulatory complex. Expression of TnC in bacteria (Chen et al, 1988;Reinach & Karlsson, 1988;Xu & HitchcockDeGregori, 1988) and the analysis of site-directed mutants (Fujimori et al, 1990;Grabarek et al, 1990;Putkey et al, 1991;Sheng et a]., 1991;Negele et al, 1992;Pearlstone et al, 1992;Silva et al, 1993) in conjunction with the determination of the crystal structure of TnC (Herzberg & James, 1985;Sundarlingam et al, 1985) has provided a better understanding of the calcium-induced conformational change in TnC (Silva & Reinach, 1991;Grabarek et al, 1992). This conformational change is responsible for the modulation of the inhibitory action of Tnl.…”