The Conformational Properties of Some Proline-Rich Fragments in Titin

 

Shiuan-Yi Huang1, Wei-Chen Lin2, Lou-Sing Kan1,2, Kuan Wang3

 

1Institute of Biochemistry, National Chung-Hsing University, Taichung, R.O.C.

2Institute of Chemistry, Academia Sinica, Taipei, R.O.C.

3National Institutes of Health, U.S.A.

 

 

Titin is a giant elastic protein in striated muscle, which is composed of tandem, repeats of about 100 residue immunoglobin domains and the PEVK modules, which was believed to play a role of the elasticity of muscle. The structure of a 28-mer PEVK repeated module (HTR12) was proposed containing three short PPII helices region within -PXXP motif (X= a.a. residue), such as a semi-rigid, rod-shaped conformation like two solid bars with a flexible rope linked each other (Ma et al., Biochemistry, 2001). In order to study the conformational properties of short PPII helix region in the 28-PEVK (HTR12) module, we designed and synthesized the three proline-rich subfragments; PR1: KPEVPPVKV, PR2: KKAPVAPPK, PR3: PEPPKEVVPE, which three sequences were divided from the 28-mer PEVK sequence. The CD and NMR were preformed the impact of the role of proline residue on the conformation of the PPII helix region in aqueous solution. These peptide fragments were displayed the characteristic PPII CD spectra (a weak positive shoulder band at 223 nm and a strong negative band near 200 nm). From temperature dependent experiment, the CD amplitude at 223 nm of three peptide showed a linear relationship at low and high temperature region, but a turning point is observed nearby 50.This turning point was not due to the cis/trans isomerization of proline residues, confirmed by the NMR NOE data. In addition, the amide proton's chemical shift with the temperature also have the similar result that in consistent with the temperature dependent CD experiment. According to our data, this phenomenon is proposed to the non-cooperative disordering of the conformation caused by destruction of the polypeptide hydration shell. Our study will facilitate the understanding of function of PEVK in titin. ( This work is supported by NSC grants and Institute of Chemistry, Academia Sinica.)