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Various types of cross-linkers with different backbone features are widely used in the study of proteins. However, it is still not clear how the cross-linkers\u2019 backbone affect their own structure and their interactions with proteins. In this study, we systematically characterized and compared methylene backbone and polyethylene glycol (PEG) backbone cross-linkers in terms of capturing protein structure and dynamics. The results indicate the cross-linker with PEG backbone have a better ability to capture the inter-domain dynamics of calmodulin, adenylate kinase, maltodextrin binding protein and dual-specificity protein phosphatase. We further conducted quantum chemical calculations and all-atom molecular dynamics simulations to analyze thermodynamic and kinetic properties of PEG backbone and methylene backbone cross-linkers. Solution nuclear magnetic resonance was employed to validate the interaction interface between proteins and cross-linkers. Our findings suggest that the polarity distribution of PEG backbone enhances the accessibility of the cross-linker to the protein surface, facilitating the capture of sites located in dynamic regions. By comprehensively benchmarking with disuccinimidyl suberate (DSS)\/bis-sulfosuccinimidyl-suberate(BS3), bis-succinimidyl-(PEG)2 revealed superior advantages in protein dynamic conformation analysis in vitro and in vivo, enabling the capture of a greater number of cross-linking sites and better modeling of protein dynamics. Furthermore, our study provides valuable guidance for the development and application of PEG backbone cross-linkers.<\/jats:p>","DOI":"10.1093\/bib\/bbae026","type":"journal-article","created":{"date-parts":[[2024,2,12]],"date-time":"2024-02-12T07:01:05Z","timestamp":1707721265000},"source":"Crossref","is-referenced-by-count":10,"title":["Enhancing protein dynamics analysis with hydrophilic polyethylene glycol cross-linkers"],"prefix":"10.1093","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-6886-3565","authenticated-orcid":false,"given":"Min","family":"Sun","sequence":"first","affiliation":[{"name":"State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Innovation Academy for Precision Measurement Science and Technology Chinese Academy of Sciences , Wuhan, Hubei 430071, China"},{"name":"University of Chinese Academy of Sciences , Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Chen","sequence":"additional","affiliation":[{"name":"CAS Key Laboratory of Separation Science for Analytical Chemistry , National Chromatographic R. & A. 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Center, Dalian Institute of Chemical Physics, , Dalian, Liaoning 116023, China"},{"name":"School of Chemistry and Material Science, University of Science and Technology of China , Hefei, Anhui 230026, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chang","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Innovation Academy for Precision Measurement Science and Technology Chinese Academy of Sciences , Wuhan, Hubei 430071, China"},{"name":"University of Chinese Academy of Sciences , Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2543-1547","authenticated-orcid":false,"given":"Lihua","family":"Zhang","sequence":"additional","affiliation":[{"name":"CAS Key Laboratory of Separation Science for Analytical Chemistry , National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, , Dalian, Liaoning 116023, China"},{"name":"Chinese Academy of Sciences , National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, , Dalian, Liaoning 116023, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maili","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Innovation Academy for Precision Measurement Science and Technology Chinese Academy of Sciences , Wuhan, Hubei 430071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukui","family":"Zhang","sequence":"additional","affiliation":[{"name":"CAS Key Laboratory of Separation Science for Analytical Chemistry , National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, , Dalian, Liaoning 116023, China"},{"name":"Chinese Academy of Sciences , National Chromatographic R. & A. 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