Exploring the dynamics and structure of PpiB in living Escherichia coli cells using electron paramagnetic resonance spectroscopy

Yasmin Ben‐ishay (1) , Yoav Barak , Akiva Feintuch , Olivier Ouari (2) , Annalisa Pierro , Elisabetta Mileo (3) , Xun‐cheng Su , Daniella Goldfarb

1 Weizmann Institute of Science [Rehovot, Israël]
2 ICR - Institut de Chimie Radicalaire
3 BIP - Bioénergétique et Ingénierie des Protéines

The combined effects of the cellular environment on proteins led to the definition of a fifth level of protein structural organization termed quinary structure. To explore the implication of potential quinary structure for globular proteins, we studied the dynamics and conformations of Escherichia coli ( E. coli ) peptidyl‐prolyl cis/trans isomerase B (PpiB) in E. coli cells. PpiB plays a major role in maturation and regulation of folded proteins by catalyzing the cis/trans 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)‐nitroxide orthogonal labeling. We probed PpiB's residue‐specific dynamics by X‐band continuous wave EPR at ambient temperatures and its structure by double electron–electron resonance (DEER) on frozen samples. PpiB was delivered to E. coli 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 E. coli , determined by Gd(III)–Gd(III) DEER measurements, as compared with solution and human HeLa cells. This suggests an increase in the number of PpiB conformations present in E. coli 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.

Domaines: Sciences du Vivant [q-bio]

Yasmin Ben‐ishay, Yoav Barak, Akiva Feintuch, Olivier Ouari, Annalisa Pierro, et al.. Exploring the dynamics and structure of PpiB in living Escherichia coli cells using electron paramagnetic resonance spectroscopy. Protein Science, 2024, 33 (3), ⟨10.1002/pro.4903⟩. ⟨hal-04466515⟩