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Title: Mapping protein-protein interactions in homodimeric CYP102A1 by crosslinking and mass spectrometry.

Authors: Felker, Dana; Zhang, Haoming; Bo, Zhiyuan; Lau, Miranda; Morishima, Yoshihiro; Schnell, Santiago; Osawa, Yoichi

Published In Biophys Chem, (2021 Jul)

Abstract: Covalent crosslinking and mass spectrometry techniques hold great potential in the study of multiprotein complexes, but a major challenge is the inability to differentiate intra- and inter- protein crosslinks in homomeric complexes. In the current study we use CYP102A1, a well-characterized homodimeric P450, to examine a subtractive method that utilizes limited crosslinking with disuccinimidyl dibutyric urea (DSBU) and isolation of the monomer, in addition to the crosslinked dimer, to identify inter-monomer crosslinks. The utility of this approach was examined with the use of MS-cleavable crosslinker DSBU and recently published cryo-EM based structures of the CYP102A1 homodimer. Of the 31 unique crosslinks found, 26 could be fit to the reported structures whereas 5 exceeded the spatial constraints. Not only did these crosslinks validate the cryo-EM structure, they point to new conformations of CYP102A1 that bring the flavins in closer proximity to the heme.

PubMed ID: 33894563 Exiting the NIEHS site

MeSH Terms: Bacterial Proteins/chemistry*; Cross-Linking Reagents/chemistry*; Cytochrome P-450 Enzyme System/chemistry*; Mass Spectrometry; Models, Molecular; NADPH-Ferrihemoprotein Reductase/chemistry*; Protein Binding

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Last Reviewed: October 02, 2024