The insertion sequence of the N2A region of titin exists in an extended structure with helical characteristics

Holly Tiffany, Kanchan Sonkar, Matthew J Gage

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The giant sarcomere protein titin is the third filament in muscle and is integral to maintaining sarcomere integrity as well as contributing to both active and passive tension. Titin is a multi-domain protein that contains regions of repeated structural elements. The N2A region sits at the boundary between the proximal Ig region of titin that is extended under low force and the PEVK region that is extended under high force. Multiple binding interactions have been associated with the N2A region and it has been proposed that this region acts as a mechanical stretch sensor. The focus of this work is a 117 amino acid portion of the N2A region (N2A-IS), which resides between the proximal Ig domains and the PEVK region. Our work has shown that the N2A-IS region is predicted to contain helical structure in the center while both termini are predicted to be disordered. Recombinantly expressed N2A-IS protein contains 13% α-helical structure, as measured via circular dichroism. Additional α-helical structure can be induced with 2,2,2-trifluoroethanol, suggesting that there is transient helical structure that might be stabilized in the context of the entire N2A region. The N2A-IS region does not exhibit any cooperativity in either thermal or chemical denaturation studies while size exclusion chromatography and Fluorescence Resonance Energy Transfer demonstrates that the N2A-IS region has an extended structure. Combined, these results lead to a model of the N2A-IS region having a helical core with extended N- and C-termini.

Original languageEnglish (US)
Pages (from-to)1-10
Number of pages10
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1865
Issue number1
DOIs
StatePublished - Jan 1 2017

Fingerprint

Connectin
Insertional Mutagenesis
Sarcomeres
Trifluoroethanol
Fluorescence Resonance Energy Transfer
Proteins
Denaturation
Size exclusion chromatography
Circular Dichroism
Gel Chromatography
Muscle
Hot Temperature
Amino Acids
Muscles
Sensors

Keywords

  • Disordered region
  • Extended structure
  • FRET
  • Secondary structure
  • Titin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

The insertion sequence of the N2A region of titin exists in an extended structure with helical characteristics. / Tiffany, Holly; Sonkar, Kanchan; Gage, Matthew J.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1865, No. 1, 01.01.2017, p. 1-10.

Research output: Contribution to journalArticle

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