Journal
BIOPHYSICAL CHEMISTRY
Volume 274, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.bpc.2021.106590
Keywords
Crosslinking; Mass spectrometry; CXL-MS; BM3; Protein-protein interactions
Funding
- National Institutes of Health [ES007062, GM077430, NS055746]
- University of Michigan's Protein Folding Disease Initiative
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Covalent crosslinking and mass spectrometry techniques show promise in studying multiprotein complexes, with a study using CYP102A1 homodimer to identify inter-monomer crosslinks through limited crosslinking and monomer isolation. By examining 31 unique crosslinks, validation of cryo-EM structures and new conformations of CYP102A1 were achieved.
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.
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