Improving Thiol Specific Labeling Efficiency on Sites with Low Solvent Accessibility by Solid Phase Labelling (SSL)

Abstract

Cell signaling is crucial for cellular interactions to occur properly and expediently. Due to the body’s numerous available responses, many receptors bind to multiple ligands, resulting in receptors’ abilities to produce various responses depending on the initial ligand to which it binds. Of the numerous receptors in the body, our research focuses on arrestin3, a scaffold protein that plays an amplifying role in signal cascades for mitogen-activated protein kinases, making it a scaffold protein. Arrestin3 has the ability to bind multiple ligands on the same site, with distinct specificity, but how this multifaceted binding occurs is currently unknown.
Fluorine-tagged NMR is widely used for protein conformational studies as it is robust, clear, quick, and consistent in determining shifts in a residue’s hydrophobic character. By labeling single-cysteine arrestin3 mutants with 19F in designated residues, the chemical shift can be determined before and after binding with MAPK kinases. These shifts signify key changes in arrestin3 conformation, constructing a clearer image of its overall structure.
Unfortunately, previous labeling methods were only effective in labeling residues on the external environment of the protein, not sites with low solvent-accessibility, and required a three-day dialysis procedure, inhibiting expedient research. Solid-state labeling (SSL) is a quick, efficient, and non-denaturing method that provides a much higher labeling efficiency to residues with low solvent accessibility as well as reduces labeling time to approximately six hours instead of three days. This method optimization has produced high-quality NMR data for residues not formerly labeled efficiently or at all.