In-Cell EPR Distance Measurements on Ubiquitin Labeled with a Rigid PyMTA-Gd(III) Tag

Abstract

Double electron–electron resonance (DEER) measures distances between spin labels attached at well-defined sites in a protein and thus has the potential to report on conformational states of proteins in cells. In this work, we evaluate the suitability of the small and rigid 4PS-PyMTA-Gd­(III) spin label for in-cell distance measurements. Three ubiquitin double mutants were labeled with 4PS-PyMTA-Gd­(III) and delivered into human HeLa cells by electroporation (EP) and hypotonic swelling (HS). Gd­(III)–Gd­(III) DEER measurements were carried out on cells frozen after different incubation times, following delivery to test the stability of the spin label inside the cell. For both delivery methods, it was possible to derive distance distributions up to 12 h after delivery, although we observed a decrease in the amount of the delivered protein with time. Surprisingly, only one mutant reported a significant change in the distance distribution with time and only for HS delivery. On the basis of in vitro exchange experiments with Mn­(II) and comparison with the same mutant labeled with BrPSPy-DO3MA-Gd­(III) and considering the presence of Mn­(II) in the cell, we hypothesized that the change occurred as a consequence of partial Gd­(III)/Mn­(II) exchange with endogenous Mn­(II). These experiments also showed that the relative Gd­(III)/Mn­(II) binding affinity depends on the labeling site in the protein, which accounts for the lack of change with the other mutants delivered under HS conditions. We conclude that 4PS-PyMTA-Gd­(III) is a good spin label for in-cell DEER for delivery by EP, but caution should be taken when HS is used.

Pulsed Dipolar EPR Spectroscopy and Metal Ions: Methodology and Biological Applications

“…Recently, first attempts to use PDS for studying protein conformational states in cell have been reported. [31][32][33]98,99,130] Although the methodology of such experiments still needs to be improved, these reports underlined the importance of Gd 3 + -based labels for in-cell PDS applications.…”

“…If a biomolecule does not contain any of these, spin centers can be introduced through exchanging intrinsic diamagnetic metal ions for paramagnetic ones, e. g., Mg 2+ for Mn 2+ , or attaching paramagnetic tags, called spin labels, via site‐directed spin labeling (SDSL) . In the latter case, nitroxides are usually used, but spin labels based on trityls, Gd 3+ , Cu 2+ , and Mn 2+[29,30] are gaining importance, especially, for in‐cell PDS applications and, in the case of trityls, also for PDS at physiological temperatures …”

“…[11][12][13][14] If a biomolecule does not contain any of these, spin centers can be introduced through exchanging intrinsic diamagnetic metal ions for paramagnetic ones, e. g., Mg 2 + for Mn 2 + , [19] or attaching paramagnetic tags, called spin labels, via site-directed spin labeling (SDSL). [20] In the latter case, nitroxides are usually used, [21] but spin labels based on trityls, [22,23] Gd 3 + , [24][25][26] Cu 2 + , [27,28] and Mn 2 + [29,30] are gaining importance, especially, for in-cell PDS applications [23,[31][32][33] and, in the case of trityls, also for PDS at physiological temperatures. [34,35] If a biomolecule contains two or more spin centers, PDS can be applied to measure the dipolar coupling between these centers, which encodes the distance and relative orientation between the spin centers and, therefore, is an important source of structural information.…”

In‐Cell EPR: Progress towards Structural Studies Inside Cells

“…Currently,s everalr esearch groups are highly active in the design of new Gd 3 + -based spin labels with optimized properties, such as 1) al inker stable in the cellulare nvironment, 2) a very high affinity fort he coordinated metal to avoid the presence of Gd 3 + ions free in the cellular suspension,3 )a short and rigid linker to decrease the width of distance distribution, [41] 4) as mallZ FS (zero field splitting) parameter in order to have the narrower central transition and thus the best signal amplitude, [42] and 5) as maller tag size. [41,43] For example, an ewly designed Gd 3 + -based tag (4-vinyl-PyMTA) wasd emonstrated to be avaluables pin label to selectively target Cys residues of ap roline-rich peptide thanks to av inyl group. In particular,t he grafting reactioni saMichael addition of the thiol group of Cys to the vinyl group of the spin label, which results in aS ÀCb ond between the label and the protein, ab onde xpected to be more resistant than aS ÀSbond towards cleavage in cellularenvironment.…”

In-cell destabilization of a homo-dimeric protein complex detected by DEER spectroscopy

“…In-cell studies using NMR and fluorescence techniques reported on protein folding and stability in cells (17,24,(34)(35)(36)(37)(38)(39)(40), structural changes of disordered proteins (41)(42)(43) and, to some extent, on protein association e and the stabilization of oligomeric protein forms (23,25,26). EPR (electron-paramagnetic resonance)-based double electron-electron resonance (DEER, also called PELDOR) spectroscopy has been suggested as an attractive alternative method to interrogate protein structures in cells (21,42,(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55). DEER provides distance distributions between two spin labels attached to well-defined positions in proteins (56) and can thus be used to probe proteins conformations and to deduce information about oligomeric protein states (57,58).…”