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Interaction of biliverdin chromophore with near-infrared fluorescent protein BphP1-FP engineered from bacterial phytochromeтезисы доклада
- Авторы: Stepanenko Olesya V., Rumyantsev K.A., Stepanenko Olga V., Kuznetsova I.M., Verkhusha V.V., Turoverov K.K.
- Сборник: Book of Abstracts. 33rd European congress on Molecular Spectroscopy
- Тезисы
- Год издания: 2016
- Место издания: Szeged, Hungary
- Первая страница: 187
- Последняя страница: 187
- Аннотация: Bacterial phytochromes (BphPs) provide an opportunity to engineer genetically encoded near-infrared (NIR) fluorescent proteins (FPs) for in vivo imaging of animals [1]. NIR light is beneficial over visible light because of its deeper penetration into mammalian tissues and lower background autofluorescence in NIR range. NIR FPs have been engineered on the basis of truncated fragments of BphPs with only PAS and GAF domains, which are minimally required for binding of biliverdin (BV) chromophore. In natural BphPs, BV is located in a cleft of the GAF domain and bound to a conserved Cys residue in the N-terminal extension of the PAS domain. Earlier we showed that in engineered NIR FP, called BphP1-FP, and its variants BV can also bind via a Cys residue introduced into a conservative -SPXH- motif of the GAF domain, resulting in two BV adducts with different chromophore π-conjugated systems and 30-35 nm blue shift of both absorbance and fluorescence [2]. Important insights into spectral characteristics of NIR FPs were obtained in studies of a set of dimeric NIR FPs, called iRFPs, and their mutants with Cys residues in either PAS (Cys15) or GAF domains (Cys256), with Cys residues in both GAF and PAS domains, and lacking the Cys residues [3]. In that work we found that the interaction of BV with dimeric NIR FPs is governed by inter-monomer and inter-domain allosteric effects. In dimeric iRFP variants without Cys15, a covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to another monomer whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers [3]. Here we performed spectral characterization of BphP1-FP and its mutants in buffered solutions and in the presence of denaturing agents. BphP1-FP contains the Cys residues (Cys15 and Cys256) in both PAS and GAF domains whereas its mutants have a Cys residue either in PAS (BphP1-FP/C256I) or in GAF domain (BphP1-FP/C15S), or no Cys residues (BphP1-FP/C15S/C256I). Absorbance and fluorescence studies of these NIR FP variants revealed that the spectral characteristics of BphP1-FP and BphP1-FP/C15S are determined by BV covalently bound to Cys256 only. This confirms the conclusions made for dimeric iRFPs [3]. Moreover, BphP1-FP demonstrated the highest stability of the protein structure and the affinity of BV binding among the studied BphP1-FP mutants. Our data indicate that the Cys15 residue in BphP1-FP induces protein stabilization.
- Добавил в систему: Степаненко Олеся Викторовна