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Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties
- Source :
- International Journal of Molecular Sciences; Volume 23; Issue 13; Pages: 7347
- Publication Year :
- 2022
-
Abstract
- Understanding the photophysical properties and stability of near-infrared fluorescent proteins (NIR FPs) based on bacterial phytochromes is of great importance for the design of efficient fluorescent probes for use in cells and in vivo. Previously, the natural ligand of NIR FPs biliverdin (BV) has been revealed to be capable of covalent binding to the inherent cysteine residue in the PAS domain (Cys15), and to the cysteine residue introduced into the GAF domain (Cys256), as well as simultaneously with these two residues. Here, based on the spectroscopic analysis of several NIR FPs with both cysteine residues in PAS and GAF domains, we show that the covalent binding of BV simultaneously with two domains is the reason for the higher quantum yield of BV fluorescence in these proteins as a result of rigid fixation of the chromophore in their chromophore-binding pocket. We demonstrate that since the attachment sites are located in different regions of the polypeptide chain forming a figure-of-eight knot, their binding to BV leads to shielding of many sites of proteolytic degradation due to additional stabilization of the entire protein structure. This makes NIR FPs with both cysteine residues in PAS and GAF domains less susceptible to cleavage by intracellular proteases.
- Subjects :
- Organic Chemistry
Biliverdine
General Medicine
bacterial phytochrome
near-infrared biomarkers
stability
proteolytic degradation
fluorescence quantum yield
biliverdin
Catalysis
Computer Science Applications
Inorganic Chemistry
Luminescent Proteins
Bacterial Proteins
Cysteine
Phytochrome
Physical and Theoretical Chemistry
Molecular Biology
Spectroscopy
Subjects
Details
- ISSN :
- 14220067
- Volume :
- 23
- Issue :
- 13
- Database :
- OpenAIRE
- Journal :
- International journal of molecular sciences
- Accession number :
- edsair.doi.dedup.....47909dcf0f75a23d4f57b048e902d440