Structural features of skeletal muscle titin aggregates

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Abstract

Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are β-sandwiches with a predominant β-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphology in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The “KCl-aggregates” according to AFM data had the form of a “sponge”-like structure, while amorphous “glycine-aggregates” of titin formed “branching” structures. Spectrofluorometry revealed the ability of titin “glycine aggregates” to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross β-structure, a reflection of ~4.6 Å, in these aggregates. These data indicate that the “glycine-aggregates” of titin are amyloid or amyloid-like. No similar structural features were found in titin “KCl-aggregates”; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.

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L. G. Bobyleva

Institute of Theoretical and Experimental Biophysics Russian Academy of Sciences

Email: ivanvikhlyantsev@gmail.com
Russian Federation, Pushchino, Moscow Region, 142290

T. A. Uryupina

Institute of Theoretical and Experimental Biophysics Russian Academy of Sciences

Email: ivanvikhlyantsev@gmail.com
Russian Federation, Pushchino, Moscow Region, 142290

N. V. Penkov

Institute of Cell Biophysics Russian Academy of Sciences

Email: ivanvikhlyantsev@gmail.com
Russian Federation, Pushchino, Moscow Region, 142290

M. A. Timchenko

Institute of Theoretical and Experimental Biophysics Russian Academy of Sciences

Email: ivanvikhlyantsev@gmail.com
Russian Federation, Pushchino, Moscow Region, 142290

A. D. Ulanova

Institute of Theoretical and Experimental Biophysics Russian Academy of Sciences

Email: ivanvikhlyantsev@gmail.com
Russian Federation, Pushchino, Moscow Region, 142290

A. G. Gabdulkhakov

Institute of Protein Research Russian Academy of Sciences

Email: ivanvikhlyantsev@gmail.com
Russian Federation, Pushchino, Moscow Region, 142290

I. M. Vikhlyantsev

Institute of Theoretical and Experimental Biophysics Russian Academy of Sciences

Author for correspondence.
Email: ivanvikhlyantsev@gmail.com
Russian Federation, Pushchino, Moscow Region, 142290

A. G. Bobylev

Institute of Theoretical and Experimental Biophysics Russian Academy of Sciences

Email: bobylev1982@gmail.com
Russian Federation, Pushchino, Moscow Region, 142290

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2. Fig. 1. SDS gel electrophoresis and Western blotting of rabbit skeletal muscle titin. a – SDS-EP of purified titin preparation (two right lanes). Left lane – rabbit soleus m. (control). Electrophoresis was performed in 7% polyacrylamide gel. Bands of actin, myosin heavy chains (MHC), nebulin and titin are indicated. b – Western blotting of titin using AB5 monoclonal antibodies. Electrophoresis was performed in 2.2% polyacrylamide gel reinforced with agarose. Left lane – rabbit soleus m. (control). Two right lanes – purified titin preparations. c – SDS-EF in 7% polyacrylamide gel of purified titin preparation (lane on the left) and Western blotting of titin using monoclonal antibody 9D10 (lane on the right). T1 – full-length titin molecules located in the sarcomere from the M-line to the Z-disk. T2 – fragments of titin-1 located in the A-disk of the sarcomere along the myosin filaments.

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3. Fig. 2. Atomic force microscopy of titin aggregates. a – Atomic force microscopy of titin aggregates in a 0.15 M glycine-KOH solution, pH 7.0; squares 50 and 10 μm2. b – Atomic force microscopy of titin aggregates in a 200 mM KCl solution, 10 mM imidazole, pH 7.0; squares 10 and 4.5 μm2. Aggregate formation time at 4 °C – 24 h.

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4. Fig. 3. a – Intensity of TT fluorescence in the presence of titin and aggregates of this protein formed over 24 h in solutions containing 0.15 M glycine-KOH, pH 7.0 (4, purple) and 200 mM KCl, 10 mM imidazole, pH 7.0 (2, green). Red (3) – intensity of TT fluorescence in the presence of the molecular form of titin; b – FTIR spectra of titin and its aggregates at 20 °C. Protein concentration is 36–42 mg/ml. Non-aggregated skeletal muscle titin (1, black). Aggregates of skeletal muscle titin formed in a solution of 200 mM KCl, 10 mM imidazole, pH 7.0 (2, red). Aggregates of skeletal muscle titin formed in 0.15 M glycine-KOH solution, pH 7.0 (3, green).

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5. Fig. 4. X-ray diffraction of titin aggregates from rabbit skeletal muscles after 24 h of aggregation. a – X-ray diffraction pattern of amorphous titin aggregates formed in a solution containing 0.15 M glycine-KOH, pH 7.0. The following reflections were found: 5.9; 4.6; 4.3; 3.8; 3.6; 3.1; 3.04; 2.97; 2.8; 2.6; 2.53; 2.43 Å. The reflection ~4.6 Å refers to the element of the cross-β-structure and characterizes the distance between the polypeptide chains; b – X-ray diffraction pattern of amorphous titin aggregates formed in a solution containing 200 mM KCl, 10 mM imidazole, pH 7.0. The following reflections were found: 18.5; 12.5; 4.1; 3.7; 2.9; 2.48 Å.

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