Use of singlet oxygen to analyze protein structure

Proteins must undergo various types of conformational changes to fulfill their biological function. Current mass spectrometry techniques are used in structural biology and can provide a variety of information about protein structure, conformational changes or specific parts of the protein involved in interaction with other biomolecules. The idea behind the integration of mass spectrometry into structural techniques is based on the labeling of the protein by a suitable chemical probe. The structure, the accessibility of the reaction sites and other factors determine the modification of the protein by the probe.

These labeling methods are usually referred to as "protein footprinting" and reflect the protein structure. The rapid reactivity of the probing agent modifies the parts of the protein accessible to the solvent and records the molecular "footprint". The probing agent can be bound to the protein either unstably (as in the hydrogen-deuterium exchange technique) or stably (e.g. by radical modification of the protein in the Fast Photochemical Oxidation of Proteins technique).

In this work we show that singlet oxygen (¹O₂) is a suitable covalent labeling agent. Singlet oxygen is generated under mild conditions with a photosensitizer and red light of 650 nm. The photosensitizer is excited by the red light and generates singlet oxygen, which immediately reacts with the protein under investigation, preferably with the amino acid residues Cys, His, Met, Trp and Tyr. We investigated various conditions of the labeling process, including buffer composition, specificity and modification of the target residues, and tested the usefulness of the data for structural mass spectrometry.