title: Detection of protein-protein interactions by proximity-driven S(N)Ar 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 S(N)Ar 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.
authors.affiliation: Department of Medicinal Chemistry, The University of Kansas , Lawrence, Kansas 66045, United States.
authors.forename: David
authors.initials: D
authors.lastname: Hymel
authors.forename: Zachary R
authors.initials: ZR
authors.lastname: Woydziak
authors.forename: Blake R
authors.initials: BR
authors.lastname: Peterson
mesh_terms: D018360:Crystallography, X-Ray; D007074:Immunoglobulin G / Q000737:chemistry; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry; D015394:Molecular Structure; D011485:Protein Binding; D013205:Staphylococcal Protein A / Q000737:chemistry
chemical_list: D007074:Immunoglobulin G; D015335:Molecular Probes; D013205:Staphylococcal Protein A; D008239:Lysine