Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2022 May 23;13:2853. doi: 10.1038/s41467-022-30304-9

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 3 — a UHPLC-MS analysis revealed the formation of a covalent bond between DIII and the inserted cyanine dye. The increase in mass was 691 m/z, which was equal to IR-783 (Cl¯ removed). b Core structures of Cl-containing and Cl-free dyes. Some cyanine/polymethine dyes do not contain the red group(s). c Cyanine dyes interact with DIII and its variants in two independent stages. In stage I, the dyes insert into the hydrophobic pocket of DIII and bind to the pocket through non-covalent bonds, resulting in increased brightness. In stage II, covalent bonds stabilize Cl-containing dyes in the pocket. The amino acid residues buried inside the pocket also play essential roles in covalent bond formation. d Full HSA bears two binding sites for cyanine dyes: one site is situated on the surface of DIa (free Cys34 is the only natural cystine containing a free thiol group) and the other is located in the hydrophobic pocket of DIIIa (covalent bonds are formed at Cys475). Lower panel: binding residue in the sequence. e Enhanced fluorescence intensity (1.3–5.9-fold) was recorded for most of the cyanine dyes bound to DIII compared with HSA or BSA. f The fluorescence enhancement is lower for dye@HSA than dye@DIII because the dye bound to DI exhibits intramolecular quenching to the fluorescently enhanced dye bound in the hydrophobic pocket of DIII. g TICT-induced NIR-I and NIR-II states of IR-783 (top) and ICG (bottom) based on DFT and TDDFT calculations. h–j Shotgun proteomics analysis revealed that Cys475 contributes to the formation of the covalent bond. MS/MS spectra (i) and fragment masses (j) of the triply charged tryptic peptide containing the dye-modified cysteine (*indicated) amino acid. The complete y series ions (y1–y8) were acquired using an Orbitrap-based mass analyzer (<0.5 ppm). These ions are labeled on the spectrum and depicted on the sequence (upper panel). The data were analyzed using MaxQuant. Posterior error probability (PEP) and Andromeda scores are presented in the right panel. Source data are provided as a Source Data file.