Studying the Cytoskeleton by Label Transfer Crosslinking: Uses and Limitations

Abstract

HAHS is a new type of photoactivated crosslinking reagent that can transfer a radiolabel from an initially labeled protein to its neighbors, due to the presence of a radiolabeled phenyl azide distal to a cleavable ester bond. HAHS has identified several protein interactions in the red blood cell cytoskeleton. However, when used to study how actin binds to integral plasma membrane proteins from Dictyostelium discoideum, only actin-actin crosslinks formed; no membrane protein was identified. Several nonradioactive phenyl azide crosslinkers gave similar results. Nonspecific labeling with a water-soluble precursor of HAHS showed that proteins varied by a factor of 20 in their susceptibility to reaction with nitrenes, with actin being the most reactive protein tested. Correspondingly, amino acids vary by a factor of >10⁵ in their reactivity, with cysteine and the aromatic residues being the most reactive. Thus, chemical reactivity of proteins, probably because of differences in exposed amino acids, may significantly bias the outcome of crosslinking experiments.