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

Authors

  • Olesya V. Stepanenko,

    1. Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia
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  • Grigory S. Bublikov,

    1. Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia
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  • Olga V. Stepanenko,

    1. Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia
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  • Daria M. Shcherbakova,

    1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA
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  • Vladislav V. Verkhusha,

    Corresponding author
    1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA
    2. Department of Biochemistry and Developmental Biology, Institute of Biomedicine, University of Helsinki, Finland
    • Correspondence

      V. V. Verkhusha, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

      Fax: +1 718 430 8996

      Tel: +1 718 430 8591

      E-mail: vladislav.verkhusha@einstein.yu.edu

      K. K. Turoverov, Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg 194064, Russia

      Fax: +7 812 2970341

      Tel: +7 812 2971957

      E-mail: kkt@incras.ru

      I. M. Kuznetsova, Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg 194064, Russia

      Fax: +7 812 2970341

      Tel: +7 812 2971957

      E-mail: imk@incras.ru

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  • Konstantin K. Turoverov,

    Corresponding author
    1. Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia
    2. St Petersburg State Polytechnical University, Russia
    • Correspondence

      V. V. Verkhusha, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

      Fax: +1 718 430 8996

      Tel: +1 718 430 8591

      E-mail: vladislav.verkhusha@einstein.yu.edu

      K. K. Turoverov, Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg 194064, Russia

      Fax: +7 812 2970341

      Tel: +7 812 2971957

      E-mail: kkt@incras.ru

      I. M. Kuznetsova, Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg 194064, Russia

      Fax: +7 812 2970341

      Tel: +7 812 2971957

      E-mail: imk@incras.ru

    Search for more papers by this author
  • Irina M. Kuznetsova

    Corresponding author
    1. Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia
    2. St Petersburg State Polytechnical University, Russia
    • Correspondence

      V. V. Verkhusha, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

      Fax: +1 718 430 8996

      Tel: +1 718 430 8591

      E-mail: vladislav.verkhusha@einstein.yu.edu

      K. K. Turoverov, Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg 194064, Russia

      Fax: +7 812 2970341

      Tel: +7 812 2971957

      E-mail: kkt@incras.ru

      I. M. Kuznetsova, Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St Petersburg 194064, Russia

      Fax: +7 812 2970341

      Tel: +7 812 2971957

      E-mail: imk@incras.ru

    Search for more papers by this author

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.

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