Detection of Protein–Protein Interactions by Proximity-Driven S_NAr Reactions of Lysine-Linked Fluorophores

Abstract

Critical protein–protein interactions are ubiquitous in biology. To provide a new method to detect these interactions, we designed and synthesized fluorinated bromopyronins as molecular probes. These electrophilic compounds rapidly react with amines via a SNAr mechanism to form modestly electrophilic aminopyronin fluorophores. To investigate whether proteins modified with aminopyronins might selectively transfer these fluorophores between proximal lysine residues at protein–protein interfaces, immunoglobulin-G (IgG) was conjugated to fluorinated pyronins and added to unlabeled Protein A (SpA) from S. aureus. Analysis by gel electrophoresis and mass spectrometry revealed transfer of this fluorophore from IgG to specific lysines of its binding partner SpA but not to bovine serum albumin (BSA) as a nonbinding control. Examination of an X-ray structure of IgG bound to SpA revealed that the fluorophore was selectively transferred between amino groups of lysines that reside within ∼10 Å at the protein–protein interface. To evaluate whether this approach could be used to identify interactions with endogenous cellular proteins, pyronin-modified Rnase A was added to crude extracts of human HeLa cells. Analysis of interacting proteins by gel electrophoresis revealed the endogenous ribonuclease inhibitor as the primary cellular target. Given that proximal lysine residues frequently reside at protein–protein interfaces, this method may facilitate identification of diverse protein–protein interactions present in complex biological matrices.

The Cohesive Metastasis Phenotype in Human Prostate Cancer

“…The ability to recover protein complexes downstream of the laminin-binding integrins (LBI) in cohesive-clusters of prostate tumors rapidly, under native conditions, and with an increased sensitivity [92], will lead to robust analysis of LBI proteome dynamics in prostate cancer metastases. Current proximity ligation assays are capable of detecting the biomolecular protein-protein interactions utilizing lysine-linked fluorophores in tissue [93]. This technological advance, coupled with the new knowledge that cohesive tumor clusters utilize adhesion molecules for aggressive dissemination, will likely be translated into predictions of drug efficacy and sensitivity.…”

Small-Molecule Fluorophores with Large Stokes Shifts: 9-Iminopyronin Analogues as Clickable Tags

“…38 We therefore assumed that pyronins substituted by nitrogen-atom containing substituents at position 9 could be excellent candidates for fluorophores that exhibit very large Stokes shifts. Wu and Burgess have proposed that an acid−base equilibrium is likely established by treating 9-(alkylamino)pyronin 9 (form A) with triethylamine as a base to form a deprotonated species B (Scheme 4).…”

“…Although only a few pyronin analogues bearing a substituted nitrogen-atom at position 9, such as alkylamino, dialkylamino, 20,35,36 or bis(trimethylsilyl)amino 37 groups, have been reported, 9-(dialkylamino)pyronins have already been used in a recent study of protein−protein interactions. 38 We therefore assumed that pyronins substituted by nitrogen-atom containing substituents at position 9 could be excellent candidates for fluorophores that exhibit very large Stokes shifts. Wu and Burgess have proposed that an acid−base equilibrium is likely established by treating 9-(alkylamino)pyronin 9 (form A) with triethylamine as a base to form a deprotonated species B (Scheme 4).…”

“…However, the origin of these large Stokes shifts has not been elucidated. Although only a few pyronin analogues bearing a substituted nitrogen-atom at position 9, such as alkylamino, dialkylamino, ,, or bis(trimethylsilyl)amino groups, have been reported, 9-(dialkylamino)pyronins have already been used in a recent study of protein–protein interactions . We therefore assumed that pyronins substituted by nitrogen-atom containing substituents at position 9 could be excellent candidates for fluorophores that exhibit very large Stokes shifts.…”

Site-Specific Turn-On Fluorescent Labeling of DNA-Interacting Protein Using Oligodeoxynucleotides That Modify Lysines To Produce 5,6-Dimethoxy 3-Methyleneisoindolin-1-one

“…DNA–protein interaction is also regulated by conformational changes in proteins that cause topological changes in the lysine residue involved in such interactions. − Fluorescent labeling is a promising method for studying the localization, structures, and functions of biomolecules. Fluorescent modification of a lysine residue of interest might afford important information about its position and functions that would help in understanding protein structure, function, and molecular mechanism. − In this study, we developed oligodeoxynucleotides (ODNs) that modify primary amines to produce 5,6-dimethoxy 3-methyleneisoindolin-1-one for fluorescent protein modification at a specific lysine residue. We have previously studied the reactivity of ODNs containing 1,4-dicarbonyls ( 1 ), which are a ring-opened form of the C4′-oxidized abasic site (OAS), to modify the DNA-interacting protein at the target lysine residue. − OAS is an oxidatively damaged DNA lesion, and we have determined in an earlier study that ODNs containing OAS modify the primary amine as a five-membered ring lactam ( 2 ) under mild conditions (Scheme ).…”