TY - JOUR

T1 - EPR spectroscopy in the study of ribosomal complexes

AU - Krumkacheva, Olesya

AU - Malygin, Alexey

AU - Graifer, Dmitri

AU - Kolokolov, Mikhail

AU - Bagryanskaya, Elena

N1 - D.G. and A.M. thank the Russian state-funded project for ICBFM SB RAS (grant number 125012300658–9); E.B. thanks the Russian Science Foundation (grant № 25–13-327). O.K. and M.K. thank the Russian Ministry of Science and Higher Education (Contract No. 075–15-2021–580).

PY - 2025/8/6

Y1 - 2025/8/6

N2 - Protein synthesis is a fundamental biological process universally mediated by ribosomes—complex ribonucleoprotein assemblies responsible for translating genetic information into functional proteins. Despite significant structural information provided by X-ray crystallography and cryo-electron microscopy (cryo-EM), certain dynamic features of ribosomal function, particularly those involving RNA conformational flexibility and transient interactions, remain challenging to characterize. Electron Paramagnetic Resonance (EPR) spectroscopy, combined with site-directed spin labeling (SDSL), has emerged as a robust complementary approach for probing structural dynamics and conformational heterogeneity in ribosomal complexes. This review summarizes recent advances in applying EPR spectroscopy, particularly pulse dipolar EPR (DEER/PELDOR), to investigate human ribosomal complexes. We discuss methodological aspects of spin-labeling strategies for mRNA, comparing various nitroxide-based labels and highlighting their specific advantages for probing ribosomal interactions. Through representative examples, we illustrate how different EPR techniques yield complementary structural information in studying ribosome-RNA interactions. Key findings include the identification of alternative mRNA conformations within ribosomal complexes, characterization of labile RNA binding sites near the mRNA entry channel, and elucidation of stabilization effects mediated by tRNA interactions. Furthermore, we demonstrate how the integration of EPR data with molecular modeling facilitates accurate interpretation of distance distributions, enabling the correlation of experimental findings with atomic-level structural models. Finally, we address current methodological limitations of EPR spectroscopy, outlining promising perspectives and anticipated advancements in this evolving field.

AB - Protein synthesis is a fundamental biological process universally mediated by ribosomes—complex ribonucleoprotein assemblies responsible for translating genetic information into functional proteins. Despite significant structural information provided by X-ray crystallography and cryo-electron microscopy (cryo-EM), certain dynamic features of ribosomal function, particularly those involving RNA conformational flexibility and transient interactions, remain challenging to characterize. Electron Paramagnetic Resonance (EPR) spectroscopy, combined with site-directed spin labeling (SDSL), has emerged as a robust complementary approach for probing structural dynamics and conformational heterogeneity in ribosomal complexes. This review summarizes recent advances in applying EPR spectroscopy, particularly pulse dipolar EPR (DEER/PELDOR), to investigate human ribosomal complexes. We discuss methodological aspects of spin-labeling strategies for mRNA, comparing various nitroxide-based labels and highlighting their specific advantages for probing ribosomal interactions. Through representative examples, we illustrate how different EPR techniques yield complementary structural information in studying ribosome-RNA interactions. Key findings include the identification of alternative mRNA conformations within ribosomal complexes, characterization of labile RNA binding sites near the mRNA entry channel, and elucidation of stabilization effects mediated by tRNA interactions. Furthermore, we demonstrate how the integration of EPR data with molecular modeling facilitates accurate interpretation of distance distributions, enabling the correlation of experimental findings with atomic-level structural models. Finally, we address current methodological limitations of EPR spectroscopy, outlining promising perspectives and anticipated advancements in this evolving field.

KW - Conformational heterogeneity

KW - DEER/PELDOR

KW - EPR spectroscopy

KW - Pulse dipolar EPR

KW - RNA

KW - Ribosome

KW - Translation

KW - mRNA dynamics

UR - https://www.mendeley.com/catalogue/c3d0207a-7806-317f-a427-6dc304bad81e/

UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=105012858290&origin=inward

U2 - 10.1007/s12551-025-01348-0

DO - 10.1007/s12551-025-01348-0

M3 - Review article

JO - Biophysical Reviews

JF - Biophysical Reviews

SN - 1867-2450

ER -