In‐Cell EPR: Progress towards Structural Studies Inside Cells

Abstract

Exploring the structure and dynamics of biomolecules in the context of their intracellular environment has become the ultimate challenge for structural biology. As the cellular environment is barely reproducible in vitro, investigation of biomolecules directly inside cells has attracted a growing interest. Among magnetic resonance approaches, site‐directed spin labeling (SDSL) coupled to electron paramagnetic resonance (EPR) spectroscopy provides competitive and advantageous features to capture protein structure and dynamics inside cells. To date, several in‐cell EPR approaches have been successfully applied to both bacterial and eukaryotic cells. In this review, the major advances of in‐cell EPR spectroscopy are summarized, as well as the challenges this approach still poses.

Nickel and GTP Modulate Helicobacter pylori UreG Structural Flexibility

“…SDSL-EPR spectroscopy is a non-destructive technique that provides details on protein structure and flexibility over a wide-range of temperatures and timescales [35][36][37][38]. Proteins can be studied in their native environment, that is in membranes, in cellular extract and also inside cells [39]. SDSL-EPR involves the grafting of a paramagnetic label, generally a thiol-specific nitroxide, on the protein of interest and the determination of the dynamic properties of the attached nitroxide by continuous wave (CW)-EPR spectroscopy [40,41].…”

Isoindoline‐Based Nitroxides as Bioresistant Spin Labels for Protein Labeling through Cysteines and Alkyne‐Bearing Noncanonical Amino Acids

“…SDSL is based on chemical selective and stable linkage of an EPR‐active species to the target. Spin labeled biological macromolecules can be studied by DEER even in the context of their native, cellular environment, as biological organisms own a limited amount of endogenous EPR‐active species, for example, manganese, copper and iron ions. Endogenous paramagnetic species are spectrally distinguishable from typical spin labels such as lanthanides, copper, trityl, or—the most commonly employed spin labels—nitroxides.…”

“…There are two key features for in cell applicability of nitroxide based spin labels: 1) the stability of the nitroxide head group against intracellular reduction, which is known to be a limiting factor in biological surroundings and 2) the attachment strategy.…”

A Two‐Armed Probe for In‐Cell DEER Measurements on Proteins**

“…The resistance of nitroxides towards reductionc an be increased through modification of the nitroxide-containingr ing, usually by attaching substituents. [18,19] Other approaches replaced the nitroxide by other radicalt ypes, such as the trityl radical; [20,21] see also the review by Bonucci et al [22] Here we focus on one such alternative, the Gd III ion, with S = 7/2, which has ab etter stability than the standard nitroxides and, especially at high magnetic field, provides high sensitivity and therefore is ag ood candidate for in-cell DEER measurements. [23][24][25] AD OTA( 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid) based Gd III complex, functionalizedwith amaleimide group,w as successfully used for in-cell DEER on ap rotein, althoughawide distance distribution was found, due to the flexibility of the linker.…”

“…The resistance of nitroxides towards reduction can be increased through modification of the nitroxide‐containing ring, usually by attaching substituents [18, 19] . Other approaches replaced the nitroxide by other radical types, such as the trityl radical; [20, 21] see also the review by Bonucci et al [22] …”