Probing conformational changes by protein surface azidation

PROJECT SUMMARY
Despite the rapid emergence of biophysical tools to detect and characterize conformational
changes in protein structure, studying protein dynamics with high sensitivity and reliability in its
native environment remains a formidable challenge. Laborious sample preparation and
requirement for special equipment present a major obstacle for democratizing these tools. Thus,
a simple yet robust platform for characterizing dynamic changes in protein conformation is highly
demanded. Using azide-containing hypervalent iodine reagents, we have developed a novel
chemoproteomic platform termed Protein Surface Azidation Mass Spectrometry (ProSurA-MS)
that detects conformational changes in proteins with unbiased chemoselectivity. Combined with
bioorthogonal chemistry, ProSurA-MS allows proteome-wide, site-specific profiling of protein
surfaces with wide coverage and reproducibility. ProSurA-MS effectively mapped conformational
changes of purified proteins upon denaturation, protein-small molecule interaction, and protein-
protein interaction. Additionally, ProSurA-MS detected structural changes in a zinc-binding protein
in whole cell lysate upon zinc depletion and measured proteome-wide azidation in live cells,
potentiating the characterization of protein dynamics in complex biological environments. The
herein proposed ProSurA-MS studies will enable i) characterization of dynamic changes in protein
conformation induced by post-translational modifications in response to oxidative stress and
monitoring of the protein dynamics of different genetic variants of a metal transporter (Aim 1), ii)
basic understanding of the chemical mechanism behind the ProSurA reaction and development
of second generation reagents with greater azidation yield and surface coverage (Aim 2), and iii)
establishment of a novel method for the identification of protein-protein interactions based on
protein surface azidation in live cells (Aim 3).

Grant Number: 5R01GM145886-05
NIH Institute/Center: NIH
Principal Investigator: Alexander Adibekian