Selective Bridging of Bis-Cysteinyl Residues by Arsonous Acid Derivatives as an Approach to the Characterization of Protein Tertiary Structures and Folding Pathways by Mass Spectrometry
✍ Scribed by H.Peter Happersberger; Michael Przybylski; Michael O. Glocker
- Publisher
- Elsevier Science
- Year
- 1998
- Tongue
- English
- Weight
- 215 KB
- Volume
- 264
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Bis
-cysteine selective modifications were successfully applied with melarsen oxide (MEL), an arsonous acid derivative, for tertiary structural studies of peptides and a model protein. The arsonous acid modified peptides and proteins were amenable to direct characterizations by mass spectrometry, e.g., direct molecular weight determinations and mass spectrometric peptide mapping that identified stoichiometry and sites of modification, respectively. Proteolytic digestion and mass spectrometric fragmentation of modified oxytocin showed that MEL-bridged peptide derivatives are structural homologues to the disulfidebonded macrocyclic peptides. Mass spectrometric analyses determined the MEL modification site in partially reduced and selectively modified bovine pancreatic trypsin inhibitor (BPTI) bridging Cys-14 and Cys-38. The BPTI ⅐ MEL derivative was resistant to proteolysis by both Lys-C and trypsin and thus represented a rigid structure like native BPTI. MEL exhibited several advantageous features such as (i) crosslinking two closely spaced thiol groups, providing detailed tertiary structure information; (ii) high solubility as monomeric ortho acid in aqueous and organic solutions; (iii) adding a relatively large mass increment to proteins upon single modification; (iv) enabling UV monitoring of the derivatization due to a strong chromophor; and (v) performing fast and specific modifications of bis-thiol groups in proteins to form stable structures without any side reactions even with a high molar excess of MEL. The investigated physical and chemical properties of MEL suggest general applicability for selective bis-thiol modifications, enabling protein structure-function studies in both soluble and membrane proteins and the study of protein-folding reactions.