Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5632, USA.
Department of Molecular & Integrative Physiology, 7744 MS II, 1137 E. Catherine St., Ann Arbor, MI 48109-5622, USA.
Biophys Chem. 2021 Jul;274:106590. doi: 10.1016/j.bpc.2021.106590. Epub 2021 Apr 20.
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.
共价交联和质谱技术在研究多蛋白复合物方面具有巨大的潜力，但一个主要的挑战是无法区分同型复合物中的内蛋白和外蛋白交联。在本研究中，我们使用 CYP102A1，一种经过充分表征的同源二聚体 P450，来检验一种减法方法，该方法利用有限的交联与二琥珀酰亚胺基二丁酸脲（DSBU），以及单体的分离，除了交联的二聚体外，来识别单体间的交联。该方法的实用性通过使用 MS 可裂解交联剂 DSBU 以及最近发表的基于 cryo-EM 的 CYP102A1 同源二聚体结构进行了检验。在发现的 31 个独特交联中，有 26 个可以拟合到报告的结构，而有 5 个超出了空间限制。这些交联不仅验证了 cryo-EM 结构，还指出了 CYP102A1 的新构象，使黄素更接近血红素。
