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

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.

Structural proteomics can be used for studying protein structures which may be difficult to examine by traditional structural biology methods such as NMR or X-ray crystallography. Crosslinking in particular is used to provide distance constraints for molecular modeling of individual proteins and protein complexes. The shortest distance constraints are most valuable for the modeling process. To be able to provide such short distance constraints, non-specific photo-reactive chemistry can be used for crosslinking reactions. However, detection of such non-specific crosslinks is difficult because the signal from any particular crosslink is low due to the broad reactivity of the crosslinking reagents. To overcome this problem, we have employed isotopic labeling of these crosslinkers. In this paper, we have demonstrated their effectiveness for studying the native α-synuclein protein structure. The non-specific reactivity, in combination with isotopic coding of these crosslinkers, allowed for the formation and detection of short-range crosslinks, targeting a variety of amino acids. These reagents may prove useful for future applications to a variety of protein structural problems. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.