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Fluorescent or affinity tags are often used as sensitive readouts for catalytic variability and specificity of E2 enzymes and their variants. Here, we examined how the size or placement of fluorescent tags compromise Ub conjugation for the E2 enzymes UbcH7, UbcH5a, and Ubc13. Large green fluorescent protein (GFP)-based fluorophores were placed at the N- or C-terminus of the E2 enzyme UbcH7 and the N-terminus of Ub, and compared with Alexa-based dyes, approximately 30 times smaller, at the same positions. While all modified proteins formed E2\u223cUb conjugates, placing a smaller Alexa dye at the C-terminus of the E2 enzyme improves the initial rate of E2\u223cUb conjugate formation by up to 10-fold compared with a larger GFP-based tag (YPet) at the N-terminus. In the case of UbcH5a, YPet labelling at its N-terminus significantly slowed formation of an E2\u223cUb conjugate and Ub unloading to the HECT domain of HUWE1 when compared to UbcH7. Overall, we find that small Alexa-based tags, especially when placed at the C-terminus of E2 enzymes, are an optimal choice for monitoring E2 ubiquitination using fluorescence-based experiments.<\/jats:p>","DOI":"10.1042\/bcj20260496","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T13:52:54Z","timestamp":1790085174000},"page":"2091-2106","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Fluorophore placement and size impact E2 ubiquitination efficiency"],"prefix":"10.1042","volume":"483","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5317-3090","authenticated-orcid":false,"given":"Jashanjot\u00a0Kaur","family":"Gill","sequence":"first","affiliation":[{"name":"Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3405-2170","authenticated-orcid":false,"given":"Rachel\u00a0E.","family":"Lacoursiere","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4685-9625","authenticated-orcid":false,"given":"Gary\u00a0S.","family":"Shaw","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2026,10,8]]},"reference":[{"key":"2026100803561827400_B1","doi-asserted-by":"publisher","first-page":"1229","DOI":"10.1016\/j.dnarep.2010.09.011","article-title":"The ubiquitous role of ubiquitin in the DNA damage response","volume":"9","author":"Al-Hakim","year":"2010","journal-title":"DNA Repair"},{"key":"2026100803561827400_B2","doi-asserted-by":"publisher","first-page":"1537","DOI":"10.1021\/cr800414e","article-title":"The multiple layers of ubiquitin-dependent cell cycle control","volume":"109","author":"Wickliffe","year":"2009","journal-title":"Chem. 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