Discriminating Changes in Protein Structure Using Tyrosine Conjugation

Abstract

Exploration of protein structure by its solvent-accessible surfaces has been widely exploited in structural biology. Amino acids most commonly targeted for covalent modification of the native folded proteins are lysine and cysteine. For the first time, the present study has leveraged ene-type chemistry targeting tyrosine residues to discriminate those solvent-exposed from those buried. We find that 4-phenyl-3H-1,2,4- triazole-3,5(4H)-dione (PTAD) can conjugate the phenolic group of tyrosine in a manner heavily influenced by the orientation and depth of the residue with respect to the protein surface. We developed a strategy to investigate protein structure by analyzing PTAD conjugations with free tyrosine, protein structure, and found it adaptable to a wide range of analytic technologies, including fluorescence, chromatography, and mass spectrometry. This study shows how the established tyrosine-specific bioconjugation chemistry can be used as an analytical tool to distinguish the conformational states of a protein where traditional structural approaches are limited. Among limitations of the traditional structural techniques, are studies of low complexity proteins, such as tyrosine rich FET proteins, with essential cellular functions in transcription and DNA repair, and mutations associated with some neurodegenerative diseases (Schwartz et al., 2015a). The N-terminal low complexity domains of FET proteins have the ability to drive the formation of non-membrane bound cellular organelles, also known as granular bodies, through a process referred to as phase separation. Low complexity proteins are typically intrinsically disordered or lacking in rigid secondary structure elements. This renders the most popular NMR and X-ray crystallography methods incapable of providing high-resolution structure data and elevates the potential for a new method of structural analysis to reveal a wealth of otherwise unattainable information.