The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.

Institute of Inherited Metabolic Disorders 1st School of Medicine Charles University 128 00 Praha 2 Czech Republic

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Conformational properties of nine purified cystathionine β-synthase mutants

Cross-talk between the catalytic core and the regulatory domain in cystathionine β-synthase: study by differential covalent labeling and computational modeling