Fluorogenic probes for monitoring peptide binding to class II MHC proteins in living cells

Abstract

A crucial step in the immune response is the binding of antigenic peptides to major histocompatibility complex (MHC) proteins. Class II MHC proteins present their bound peptides to CD4(+) T cells, thereby helping to activate both the humoral and the cellular arms of the adaptive immune response. Peptide loading onto class II MHC proteins is regulated temporally, spatially and developmentally in antigen-presenting cells. To help visualize these processes, we have developed a series of novel fluorogenic probes that incorporate the environment-sensitive amino acid analogs 6-N,N-dimethylamino-2-3-naphthalimidoalanine and 4-N,N-dimethylaminophthalimidoalanine. Upon binding to class II MHC proteins these fluorophores show large changes in emission spectra, quantum yield and fluorescence lifetime. Peptides incorporating these fluorophores bind specifically to class II MHC proteins on antigen-presenting cells and can be used to follow peptide binding in vivo. Using these probes we have tracked a developmentally regulated cell-surface peptide-binding activity in primary human monocyte-derived dendritic cells.

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

“…32 peptide-bound HLA-DR protein was also determined, confirming the occupation of the 4-DAPA side chain in the expected hydrophobic pocket. 38 These phthalimide and naphthalimide-based FlAAs have also been incorporated into peptides recognizing PDZ and SH2 domains revealing their versatility in diverse applications. [39][40][41][42] The success of the dimethylaminonaphthalimide family led to their further exploration as environment-sensitive FlAAs.…”

Increased Structural Flexibility at the Active Site of a Fluorophore-conjugated β-Lactamase Distinctively Impacts Its Binding toward Diverse Cephalosporin Antibiotics

“…This fluorophore has been applied to probe the structural dynamics of membrane lipids (11) and ion channels (12), and to detect the binding of peptide ligands to SH2 domains (9) and Class II MHC proteins (13). We examined several fluorophores and report here one such engineered ␤-lactamase, PenP-E166Cb, which is based on the Class A ␤-lactamase PenP from Bacillus licheniformis.…”

“…For our design we decided to use the naphthalene-type fluorophore molecules because of their exceptional solvachromatic properties that can reflect the polarity of the local environmental at the molecule level, as well as their relative small size comparable to the amino acid, tryptophan, to allow them to be positioned close to the protein structure (7)(8)(9)(10)(11)(12)(13). This fluorophore has been applied to probe the structural dynamics of membrane lipids (11) and ion channels (12), and to detect the binding of peptide ligands to SH2 domains (9) and Class II MHC proteins (13).…”

Überresponsive Peptid‐basierte Sensoren von Signalproteinen

“…Eine Familie von solvatochromen Dimethylaminophthalimid-Fluorophoren (4-DMAP, 4-DMN und 6-DMN; Schema 1) wurde eingesetzt, um SH2- [9] und 14-3-3-Domänen [10] sowie Calmodulin- [11] und Klasse-II-MHCProteine zu untersuchen. [12] Struktur-und KonsensussequenzInformationen wurden herangezogen, um die Schlüsselstellen der Wechselwirkung zwischen einem Peptid und seiner Zielproteindomäne zu identifizieren. Besonders ein hydrophober aromatischer Rest auf einem Peptid, der innerhalb einer lipophilen Tasche eingebettet wird, sobald er mit seinem Protein bindenden Partner wechselwirkt, ist ein idealer Kandidat für den Austausch gegen einen solvatochromen Fluorophor.…”