Determining protein structure with tyrosine bioconjugation

Abstract

A hallmark of many neurodegenerative diseases like Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Frontal Temporal Lobar Dementia (FTLD) is the formation of misfolded proteins into aggregates in the neuronal cells. These proteins often have intrinsically disordered regions which are enriched in a low complexity of amino acids such as Serine, Glycine, Glutamic acid and Tyrosine. Disordered proteins are challenging to study with common techniques such as x‐ray crystallography and NMR. Furthermore, common biorthogonal chemical reactions which mostly target lysine and cysteine residues cannot be used to determine the protein fold due to the low variety of amino acid composition in these proteins. However, a unique characteristic of several disordered proteins involved in neurodegenerative disease is their enrichment for tyrosines. Here we have developed an approach to assess tyrosine accessibility as a measure of levels in native vs misfolded forms of the proteins.To measure tyrosine accessibility we employed the ability of 4‐Phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) to conjugate to tyrosine side‐chains. We assessed the targeted labeling of tyrosine’s for peptides, proteins, and cells. We have used this chemical approach to observe the transition of Bovine Serum Albumin (BSA), a well‐characterized and folded protein, to an unfolded state. We found that we could assess the local changes to protein structure by using LC‐MS/MS to quantify levels at each tyrosine position. We could confirm changes using fluorescent detection through click chemistry. Our long‐term goal is combining this approach and proteomics to quantify changes in cells to protein folding in comparable cellular states, such as disease and non‐diseased tissues.Support or Funding InformationNational Institute of Health, American Cancer Society