Detection of Protein鈥揚rotein Interactions by Proximity-Driven SNAr Reactions of Lysine-Linked Fluorophores
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- 作者：David Hymel ; Zachary R. Woydziak ; Blake R. Peterson
- 刊名：Journal of the American Chemical Society
- 出版年：2014
- 出版时间：April 9, 2014
- 年：2014
- 卷：136
- 期：14
- 页码：5241-5244
- 全文大小：417K
- 年卷期：v.136,no.14(April 9, 2014)
- ISSN：1520-5126
文摘
Critical protein鈥損rotein 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鈥損rotein 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 脜 at the protein鈥損rotein 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鈥損rotein interfaces, this method may facilitate identification of diverse protein鈥損rotein interactions present in complex biological matrices.