A knot in the protein structure - Probing the near-infrared fluorescent protein iRFP designed from a bacterial phytochrome

Olesya V. Stepanenko, Grigory S. Bublikov, Olga V. Stepanenko, Daria Shcherbakova, Vladislav Verkhusha, Konstantin K. Turoverov, Irina M. Kuznetsova

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The possibility of engineering near-infrared fluorescent proteins and biosensors from bacterial phytochrome photoreceptors (BphPs) has led to substantial interest in this family of proteins. The near-infrared fluorescent proteins have allowed non-invasive bio-imaging of deep tissues and whole organs in living animals. BphPs and derived near-infrared fluorescent proteins contain a structural element, called a knot, in their polypeptide chains. The formation of knot structures in proteins was refuted for a long time. Here, we studied the denaturation and renaturation processes of the near-infrared fluorescent probe iRFP, engineered from RpBphP2, which utilizes a heme-derived tetrapyrrole compound biliverdin as a chromophore. iRFP contains a unique figure-of-eight knot. The denaturation and renaturation curves of the iRFP apoform coincided well, suggesting efficient refolding. However, the iRFP holoform exhibited irreversible unfolding and aggregation associated with the bound chromophore. The knot structure in the apoform did not prevent subsequent binding of biliverdin, resulting in the functional iRFP holoform. We suggest that the irreversibility of protein unfolding is caused by post-translational protein modifications, such as chromophore binding, rather than the presence of the knot. These results are essential for future design of BphP-based near-infrared probes, and add important features to our knowledge of protein folding. Near-infrared fluorescent biomarker iRFP engineered from RpBphP2 which utilizes biliverdin as a chromophore contains a unique figure-of-eight knot. The iRFP holoform exhibited the irreversible unfolding and aggregation, while the denaturation of the iRFP apoform is reversible. The observed irreversibility of unfolding of iRFP holoform is caused by post-translational modifications rather than the knot.

Original languageEnglish (US)
Pages (from-to)2284-2298
Number of pages15
JournalFEBS Journal
Volume281
Issue number9
DOIs
StatePublished - 2014

Fingerprint

Phytochrome
Biliverdine
Chromophores
Infrared radiation
Denaturation
Post Translational Protein Processing
Proteins
Tetrapyrroles
Protein Unfolding
Bacterial Proteins
Protein Folding
Biosensing Techniques
Agglomeration
Heme
Fluorescent Dyes
Protein folding
Biomarkers
Biosensors
Peptides
Animals

Keywords

  • bacteriophytochrome
  • biliverdin
  • iRFP
  • knot
  • RpBphP2

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

A knot in the protein structure - Probing the near-infrared fluorescent protein iRFP designed from a bacterial phytochrome. / Stepanenko, Olesya V.; Bublikov, Grigory S.; Stepanenko, Olga V.; Shcherbakova, Daria; Verkhusha, Vladislav; Turoverov, Konstantin K.; Kuznetsova, Irina M.

In: FEBS Journal, Vol. 281, No. 9, 2014, p. 2284-2298.

Research output: Contribution to journalArticle

Stepanenko, Olesya V. ; Bublikov, Grigory S. ; Stepanenko, Olga V. ; Shcherbakova, Daria ; Verkhusha, Vladislav ; Turoverov, Konstantin K. ; Kuznetsova, Irina M. / A knot in the protein structure - Probing the near-infrared fluorescent protein iRFP designed from a bacterial phytochrome. In: FEBS Journal. 2014 ; Vol. 281, No. 9. pp. 2284-2298.
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