TY - JOUR
T1 - A knot in the protein structure - Probing the near-infrared fluorescent protein iRFP designed from a bacterial phytochrome
AU - Stepanenko, Olesya V.
AU - Bublikov, Grigory S.
AU - Stepanenko, Olga V.
AU - Shcherbakova, Daria M.
AU - Verkhusha, Vladislav V.
AU - Turoverov, Konstantin K.
AU - Kuznetsova, Irina M.
PY - 2014/5
Y1 - 2014/5
N2 - 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.
AB - 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.
KW - RpBphP2
KW - bacteriophytochrome
KW - biliverdin
KW - iRFP
KW - knot
UR - http://www.scopus.com/inward/record.url?scp=84899904083&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84899904083&partnerID=8YFLogxK
U2 - 10.1111/febs.12781
DO - 10.1111/febs.12781
M3 - Article
C2 - 24628916
AN - SCOPUS:84899904083
SN - 1742-464X
VL - 281
SP - 2284
EP - 2298
JO - FEBS Journal
JF - FEBS Journal
IS - 9
ER -