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Fast cross-linking by DOPA2 promotes the capturing of a stereospecific protein complex over nonspecific encounter complexes

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Biophysics Reports 2022年第Z1期8卷 239-252页

作者： Jian-Hua Wang Zhou Gong Xu Dong Shu-Qun Liu Yu-Liang Tang Xiaoguang Lei Chun Tang Meng-Qiu Dong National Institute of Biological Sciences (NIBS) Tsinghua Institute of Multidisciplinary Biomedical Research Tsinghua University State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics Innovation Academy for Precision Measurement Science and TechnologyChinese Academy of Sciences State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan Yunnan University Beijing National Laboratory for Molecular Sciences College of Chemistry and Molecular EngineeringCenter for Quantitative BiologyPeking-Tsinghua Center for Life SciencePeking University

Transient and weak protein–protein interactions are essential to many biochemical reactions, yet are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry analysis(CXMS) provides a powerful tool in the analysis of such interactions. Central to this technology are chemical cross-linkers. Here, using two transient heterodimeric complexes EIN/HPr and EIIAGlc/EIIBGlc as our model systems, we evaluated the effects of two amine-specific homo-bifunctional cross-linkers with different reactivities. We showed previously that DOPA2(di-ortho-phthalaldehyde with a di-ethylene glycol spacer arm) cross-links proteins 60–120 times faster than DSS(disuccinimidyl suberate). We found that though most of the intermolecular cross-links of either cross-linker are consistent with the encounter complexes(ECs), an ensemble of short-lived binding intermediates,more DOPA2 intermolecular cross-links could be assigned to the stereospecific complex(SC), the final lowest-energy conformational state for the two interacting proteins. Our finding suggests that faster cross-linking captures the SC more effectively and cross-linkers of different reactivities potentially probe protein–protein interaction dynamics across multiple timescales.

关键词： Mass spectrometry Chemical cross-linking Cross-linker Transient protein–protein interaction Encounter complexes stereospecific complex DOPA2 DSS

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