Troponin-C-mediated calcium-sensitive changes in the conformation of troponin I detected by pyrene excimer fluorescence.

G M Strasburg,P C Leavis,J Gergely

doi:10.1016/s0021-9258(18)89741-1

Abstract

Troponin I (TnI) from rabbit white skeletal muscle was labeled at cysteines 48 and 64 with the fluorescent reagent N-(1-pyrene)maleimide. The fluorescence spectra of pyrene-labeled TnI (pyr-TnI) exhibit peaks characteristic of pyrene in its monomeric form and an additional peak resulting from formation of excited dimers (excimers), indicating that the labeled cysteines are close together. Formation of a pyr-TnI-TnC complex in the absence of Ca2+ has little effect on the spectrum, but when Ca2+ is bound to the low-affinity sites of TnC there is a substantial decrease in excimer and a corresponding increase in monomer fluorescence. The involvement of the low-affinity sites in the Ca2+-induced effect is consistent with the fact that Mg2+ has no effect on pyrene fluorescence. On rapid mixing of the pyr-TnI-TnC complex with Ca2+ in a stopped-flow apparatus, most of the excimer decrease is complete within the instrumental dead time, indicating a rate constant k greater than 350 s-1, which is comparable to that of the conformational change in TnC resulting from Ca2+ binding to the low-affinity sites. Rapid mixing of the Mg2-TnC-pyr-TnI complex with Ca2+ yields similar results, suggesting that the type of metal ion present at the high-affinity sites has little, if any, effect on the probe. It has been suggested previously that Cys 48 and 64 are located in a TnT-binding region of TnI (Chong P.C.S. and Hodges, R.S. (1982) J. Biol. Chem. 255, 3757). Our results suggest that a Ca2+-induced structural change in the TnI-binding region of TnC could be transmitted to TnT by affecting the TnT-binding region of TnI as part of the chain of events in the regulation of muscle contraction.

Highlights

Troponin I (TnI) from rabbit white skeletal muscle shown to bind to tropomyosin
The fluorescence complex inhibition of activation of myosin ATPase by actin spectra of pyrene-labeled TnIexhibit peaks characteristic of pyrene inits monomeric form and an additional peak resulting from formation of excited dimers,indicating that thelabeled cysteines are close together
Our results suggest that a Ca2+-induced came from the workof Horwitz et al [14] showing that a structural change in the TnI-binding region of TnC biologically active troponin complex can only be formed if the couldbe transmitted to TnT by affecting the TnT- sulfhydryl groups of TnI are keptfully reduced

Summary

The spectrumof labeled TnI complexed with TnC shows

CM-TnI was rechromatographed on a Sephadex G-25column equilibrated with 0.5 M KCl, 25 mM MES, pH 6.0.Solid GdmCl was added to a final concentration of 5 M to thepooled fractions containing CM-TnI (1-3 mg/ml), followed by addition of N-(I-pyrene)maleimide dissolved in dimethyl formamide (1mg/ml) in a 20:l molar ratio. DABMA, dissolved in dimethyl formamide (1mg/ml), was added to a 1O:l molar ratio to thteroponin complex, andafter 4 h a t room temperature,the reaction was quenched with dithiothreitol. Circular dichroism measurements were carried out on labeled and unlabeled TnI (0.2mg/ml of protein concentration) in solutions containing 0.15 M KC1, 25 mM Pipes buffer (pH 6.8),and 0.5 mM dithiothreitol. In another setof experiments, pyrll-TnI-TnC, dissolved in Caz+-freebuffer (0.1M KCI, 25 mM HEPES, pH7.5,1 mM EGTA), was mixed with an equal volume ofCa2' buffer HEPES, pH7.5,0.6mM CaCI2).Excimer fluorescence was monitored using a Corning 0-72filter to cutoff monomer fluorescence

RESULTS

ATPase activity

DISCUSSION