Dynamic Protein-Protein Complexes: How Alternative Interactions Create Ensembles and How Solution NMR and MD Simulations can Characterize them

Abstract

Protein unfolding, disassembly, and aggregation underlie many diseases, but detailed study of these processes in intact cells has been limited. Cysteine Shotgun labeling utilizes cell-permeable fluorescent dyes to label exposed cysteine residues. We applied it to study protein structure changes in response to mechanical stress on red blood cell ghosts in live mice and in whole-cell lysates in native versus urea-denaturing conditions. Labeling rate constants are calculated for any given Cys site by normalizing the protein labeling kinetics to the rapid labeling under denaturing conditions. Proteins can be identified and further analyzed by mass spectrometry to pinpoint specific, susceptible domains involved. A number of proteins contain cys with a wide variety of rate constants. This study focuses on human and mouse spectrin, Filamin A and B, Talin, and pyruvate kinase. These various proteins contain many cysteine-rich domains and have been amenable to studying by this in-cell technique. Results are confirmed by studies of purified protein.