Chemical Tagging with tert-Butyl and Trimethylsilyl Groups for Measuring Intermolecular Nuclear Overhauser Effects in a Large Protein-Ligand Complex

Abstract

Intermolecular 1 H-1 H nuclear Overhauser effects (NOE) present a powerful tool to assess contacts between proteins and binding partners, but are difficult to identify for complexes of high molecular weight. This report shows that intermolecular NOEs can readily be observed following chemical labeling with tert-butyl or trimethylsilyl (TMS) groups. Proteins can be furnished with tert-butyl or TMS groups site-specifically using genetically encoded unnatural amino acids or by chemical modification of single cysteine residues. No isotope labeling is required. The approach is demonstrated with the 95 kDa complex between tetrameric E. coli single-stranded DNA binding protein (SSB) and single-stranded DNA. Show more