Stabilizing Nitroxide Spin Labels for Structural and Conformational Studies of Biomolecules by Maleimide Treatment

Abstract

Nitroxide (NO) spin radicals are effective in characterizing structures, interactions and dynamics of biomolecules. The EPR applications in cell lysates or intracellular milieu require stable spin labels, but NO radicals are unstable in such conditions. We showed that the destabilization of NO radicals in cell lysates or even in cells is caused by NADPH/NADH related enzymes, but not by the commonly believed reducing reagents such as GSH. Maleimide stabilizes the NO radicals in the cell lysates by consumption of the NADPH/NADH that are essential for the enzymes involved in destabilizing NO radicals, instead of serving as the solo thiol scavenger. The maleimide treatment retains the crowding properties of the intracellular components and allows to perform long‐time EPR measurements of NO labeled biomolecules close to the intracellular conditions. The strategy of maleimide treatment on cell lysates for the EPR applications has been demonstrated on double electron‐electron resonance (DEER) measurements on a number of NO labeled protein samples. The method opens a broad application range for the NO labeled biomolecules by EPR in conditions that resemble the intracellular milieu.

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

“…The spectra did not reveal the same changes observed in the cell, yet they did exhibit a significant contribution of a slow‐motion component. In addition, we observed a rapid decrease in the EPR signal as a function of time due to the nitroxide reduction, at a rate much faster than in the cell, as reported earlier for another protein (Bleicken et al, 2019 ; Jagtap et al, 2015 ; Karthikeyan et al, 2018 ; Wang, Zhang, et al, 2023 ). By normalizing the spectra to allow for line‐shape comparison, we detected minimal changes in the line shape with time and the decay rates of the fast and slow motion components were similar (Figure S8 ).…”

“…The majority of in‐cell EPR structural studies of proteins and nucleic acids have been followed by DEER distance measurements using nitroxide (Azarkh et al, 2013 ; Azarkh, Okle, Eyring, et al, 2011 ; Azarkh, Okle, Singh, et al, 2011 ; Cattani et al, 2017 ; Collauto et al, 2020 ; Igarashi et al, 2010 ; Joseph et al, 2015 , 2019 ; Karthikeyan et al, 2018 ; Krstić et al, 2011 ; Singewald et al, 2019 ), Gadolinium (Gd(III)) (Azarkh et al, 2019 ; Dalaloyan et al, 2019 ; Galazzo et al, 2022 ; Martorana et al, 2014 ; Qi et al, 2014 ; Yang et al, 2017 , 2018 ), and trityl (Fleck et al, 2020 ; Jassoy et al, 2017 ; Yang, Pan, et al, 2020 ) spin label pairs. Owing to the sensitivity of nitroxide spin labels to cellular reduction (Azarkh, Okle, Eyring, et al, 2011 ; Krstić et al, 2011 ; Wang, Zhang, et al, 2023 ), efforts have been made to design and synthesize nitroxide spin labels that are reduction resistant (Bleicken et al, 2019 ; Collauto et al, 2020 ; Jagtap et al, 2015 ; Karthikeyan et al, 2018 ) concurrently to the development of delivery methods that rely on a minimal time for cell recovery for E. coli (Pierro et al, 2022 ; Torricella et al, 2021 ). Moreover, endeavors to achieve in‐cell cytosolic labeling are under development (Jana et al, 2023 ; Kugele et al, 2021 ; Schmidt et al, 2014 ; Widder et al, 2020 ).…”

“…This includes nitroxide‐Gd(III) orthogonally labeled PpiB generated via the introduction of an unnatural amino acid (UAA) residue conjugated to a Gd(III) label (Abdelkader et al, 2015 ; Garbuio et al, 2013 ) and a genetically encoded cysteine residue attached to a shielded nitroxide label termed M‐TETPO (Karthikeyan et al, 2018 ) (Figure 1 ). M‐TETPO has been shown to be less prone to cellular reduction compared with the standard nitroxide spin labels (Karthikeyan et al, 2018 ; Wang, Zhang, et al, 2023 ). In addition, singly and doubly nitroxide‐labeled PpiB variants, as well as a doubly labeled Gd(III) one, were used for control and comparison purposes.…”