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To explore the implication of potential quinary structure for globular proteins, we studied the dynamics and conformations of <jats:italic>Escherichia coli<\/jats:italic> (<jats:italic>E. coli<\/jats:italic>) peptidyl\u2010prolyl <jats:italic>cis\/trans<\/jats:italic> isomerase B (PpiB) in <jats:italic>E. coli<\/jats:italic> cells. PpiB plays a major role in maturation and regulation of folded proteins by catalyzing the <jats:italic>cis\/trans<\/jats:italic> isomerization of the proline imidic peptide bond. We applied electron paramagnetic resonance (EPR) techniques, utilizing both Gadolinium (Gd(III)) and nitroxide spin labels. In addition to using standard spin labeling approaches with genetically engineered cysteines, we incorporated an unnatural amino acid to achieve Gd(III)\u2010nitroxide orthogonal labeling. We probed PpiB's residue\u2010specific dynamics by X\u2010band continuous wave EPR at ambient temperatures and its structure by double electron\u2013electron resonance (DEER) on frozen samples. PpiB was delivered to <jats:italic>E. coli<\/jats:italic> cells by electroporation. We report a significant decrease in the dynamics induced by the cellular environment for two chosen labeling positions. These changes could not be reproduced by adding crowding agents and cell extracts. Concomitantly, we report a broadening of the distance distribution in <jats:italic>E. coli<\/jats:italic>, determined by Gd(III)\u2013Gd(III) DEER measurements, as compared with solution and human HeLa cells. This suggests an increase in the number of PpiB conformations present in <jats:italic>E. coli<\/jats:italic> cells, possibly due to interactions with other cell components, which also contributes to the reduction in mobility and suggests the presence of a quinary structure.<\/jats:p>","DOI":"10.1002\/pro.4903","type":"journal-article","created":{"date-parts":[[2024,2,15]],"date-time":"2024-02-15T13:39:13Z","timestamp":1708004353000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Exploring the dynamics and structure of <scp>PpiB<\/scp> in living <i>Escherichia\u00a0coli<\/i> cells using electron paramagnetic resonance spectroscopy"],"prefix":"10.1002","volume":"33","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4363-5319","authenticated-orcid":false,"given":"Yasmin","family":"Ben\u2010Ishay","sequence":"first","affiliation":[{"name":"Department of Chemical and Biological Physics Weizmann Institute of Science  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