Ca2+-dependent photocrosslinking of tropomyosin residue 146 to residues 157-163 in the C-terminal domain of troponin I in reconstituted skeletal muscle thin filaments

Abstract

The Ca(2+)-dependent interaction of troponin I (TnI) with actin.tropomyosin (Tm) in muscle thin filaments is a critical step in the regulation of muscle contraction. Previous studies have suggested that, in the absence of Ca(2+), TnI interacts with Tm and actin in reconstituted muscle thin filaments, maintaining Tm at the outer domain of actin and blocking myosin-actin interaction. To obtain direct evidence for this Tm-TnI interaction, we performed photochemical crosslinking studies using Tm labeled with 4-maleimidobenzophenone at position 146 or 174 (Tm*146 or Tm*174, respectively), reconstituted with actin and troponin [composed of TnI, troponin T (TnT), and troponin C] or with actin and TnI. After near-UV irradiation, SDS gels of the Tm*146-containing thin filament showed three new high-molecular-weight bands determined to be crosslinked products Tm*146-TnI, Tm*146-troponin C, and Tm*146-TnT using fluorescence-labeled TnI, mass spectrometry, and Western blot analysis. While Tm*146-TnI was produced only in the absence of Ca(2+), the production of other crosslinked species did not show Ca(2+) dependence. Tm*174 mainly crosslinked to TnT. In the absence of actin, a similar crosslinking pattern was obtained with a much lower yield. A tryptic peptide from Tm*146-TnI with a molecular mass of 2601.2 Da that was not present in the tryptic peptides of Tm*146 or TnI was identified using HPLC and matrix-assisted laser desorption/ionization time-of-flight. This was shown, using absorption and fluorescence spectroscopy, to be the 4-maleimidobenzophenone-labeled peptide from Tm crosslinked to TnI peptide 157-163. These data, which show that a region in the C-terminal domain of TnI interacts with Tm in the absence of Ca(2+), support the hypothesis that a TnI-Tm interaction maintains Tm at the outer domain of actin and will help efforts to localize troponin in actin.Tm muscle thin filaments.

Fig. 1. Products of photolysis of Tm*174 and of Tm*146 in actin•Tm*•Tn. Identification of a Ca²⁺-dependent Tm-TnI crosslink

A, Tm*174 and B, Tm*146; both photolyzed in actin•Tm*•Tn, and in the presence of 2 mM Ca²⁺ (+) or in the presence of 2 mM EGTA (-). C, Tm*146 and TMR-labeled TnI photolyzed in actin•Tm*•Tn-TMR in the presence of 2mM Ca²⁺ (+) or in the presence of 2 mM EGTA (-). D. Tm*146 photolyzed in actin•Tm*•TnI. Note that Tm*146 crosslinks to TnI in actin•Tm*•Tn in the absence of Ca²⁺ (B and C, -) but not in the presence of Ca²⁺ (B and C, +), and Tm*146 crosslinks to TnI in the absence of TnC and TnT (D) . Conditions: 4 μM actin, 0.5 μM Tm, 0.5 μM Tn (or TnI) in 50 mM NaCl, 5 mM MgCl₂, 10 mM Hepes buffer, pH 7.5.

Fig. 2. Crosslinking of Tm*146 with Tn in the presence and absence of actin

Tm*146•Tn or actin•Tm*146•Tn was photolyzed under the same conditions as Fig. 1. Note that crosslinked bands at similar mobilitIes were formed in the absence of actin as well as in its presence.

Fig. 3. Identification of Tm*146-TnC and Tm*146-TnT crosslinked bands

A and B, Photolyzed samples of Tm*146 in actin•Tm*•Tn in the absence (-) and presence of Ca²⁺ (+). A, no EDTA in gel; B, EDTA (2 mM) in gel. Note the decreased mobility of TnC and Tm*146-TnC when EDTA is present in gel. C, Western blot of Tm*146-TnT using TnT antibody. Note that Tm*146 crosslinks to TnC and to TnT independent of Ca²⁺. Same conditions as Fig. 1.

Fig. 4. Effect of myosin-S1 binding to actin on the Tm*146-TnI crosslink produced in the absence of Ca²⁺

Photolysis at increasing ratios of S1/actin in the absence of Ca²⁺ and ATP, using TMR-labeled TnI in Tn. Left lane, Coomassie stained at 0 S1; Right lanes, fluorescence at increasing ratios of S1/actin. Conditions as for Fig. 1. Note the loss of crosslinks at low S1 saturation of actin.

Fig. 5. Separation of thin filament proteins before and after photolysis of actin•Tm*146•Tn using reversed phase HPLC (C4 column)

The species were identified by the molecular weights of the fractions with MALDI-TOF (see Table 1). H₂O–CH₃CN gradient in 0.1%TFA was used with increasing CH₃CN (%B). Note that Tm+Tm* is a mixture of unlabeled and labeled Tm.

Fig. 6. MALDI-TOF spectra of in-gel trypsin digests of Tm*, TnI and Tm*146-TnI

Fractions containing Tm*146-TnI from the C4 HPLC column (Fig. 4) were subjected to SDS polyacrylamide gel electrophoresis. Excised Tm*, TnI and Tm*146-TnI gel pieces were then exhaustively digested with trypsin. Note that a new peptide of 2601.9 Da was present in the crosslinked mixture (marked with an asterisk).

Fig. 7. Analysis of purified Tm*146-TnI tryptic peptides

1. Separation of trypsin digested Tm*146-TnI with reverse phase HPLC (C18 column) using a H₂O–CH₃CN gradient with increasing CH₃CN (%B) as indicated. Absorption was monitored at 280 nm. 2. Absorption spectra of tryptic peptides (fractions A and B). Note a strong absorption at 260 nm for fraction B due to presence of benzophenone. 3. Fluorescence emission spectra of fractions A and B compared to L-tryptophan in buffer excited at 280 nm. The presence of tryptophan and benzophenone verifies that the crosslinked peptide is B.