Isotopically-coded short-range hetero-bifunctional photo-reactive crosslinkers for studying protein structure.

Abstract

The resolution and the fidelity of a protein structural model, constructed using crosslinking data, is dependent on the crosslinking distance constraints. Most of the popular amine-reactive NHS-ester crosslinkers are limited in their capacity to provide short distance constraints because of the rarity of lysine residues occurring in close proximity in the protein structure. To solve this problem, hetero-bifunctional crosslinkers containing both a photo-reactive functional group and an NHS-ester group can be used to enable non-specific crosslinking within the proximity of these lysine residues. Here we develop three such isotopically-coded hetero-bifunctional photo-reactive crosslinkers, bearing azido, diazirine or benzophenone photo-reactive groups (azido-benzoic-acid-succinimide (ABAS)-(12)C6/(13)C6, succinimidyl-diazirine (SDA)-(12)C5/(13)C5, and carboxy-benzophenone-succinimide (CBS)-(12)C6/(13)C6, respectively). These crosslinkers were validated using several model proteins/peptides and were then applied to study the structure of the native α-synuclein protein. In that case the ABAS crosslinker proved to be the most suitable, with 10 crosslinks being found in the native α-synuclein structure.