Gd(III)-PyMTA Label Is Suitable for In-Cell EPR

Abstract

Distance measurement in the nanometer range by electron paramagnetic resonance spectroscopy (EPR) in combination with site-directed spin labeling is a very powerful tool to monitor the structure and dynamics of biomacromolecules in their natural environment. However, in-cell application is hampered by the short lifetime of the commonly used nitroxide spin labels in the reducing milieu inside a cell. Here, we demonstrate that the Gd(III) based spin label Gd-PyMTA is suitable for in-cell EPR. Gd-PyMTA turned out to be cell compatible and was proven to be inert in in-cell extracts of Xenopus laevis oocytes at 18 °C for more than 24 h. The proline rich peptide H-AP10CP10CP10-NH2 was site-directedly spin labeled with Gd-PyMTA at both cysteine moieties. The resulting peptide, H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2, as well as the model compound Gd-spacer-Gd, which consists of a spacer of well-known stiffness, were microinjected into Xenopus laevis oocytes, and the Gd(III)-Gd(III) distances were determined by double electron-electron resonance (DEER) spectroscopy. To analyze the intracellular peptide conformation, a rotamer library was set up to take the conformational flexibility of the tether between the Gd(III) ion and the Cα of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2 is inserted into cell membranes, coinciding with a conformational change of the oligoproline from a PPII into a PPI helix.

Gd3+–Gd3+distances exceeding 3 nm determined by very high frequency continuous wave electron paramagnetic resonance

“…Furthermore, Gd-based spin labels have been shown to be much more stable than nitroxide radicals in reducing environments, allowing for in-cell applications. 41–44 …”

Gd(III)–Gd(III) distance measurements with chirp pump pulses

“…Distance measurements involving Gd spin labels have been successfully demonstrated on a number of systems, including model compounds, peptides, nanoparticles, proteins and DNAs [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Moreover, a promising biochemical property of Gd spin labels is the stability under reducing conditions, which recently enabled Gd-Gd distance measurements of peptides and proteins embedded in cellular environments [22,23]. Because Gd(III) ions are in an S = 7/2 high spin state, high magnetic fields above 1 T are required to diminish unwanted contributions from the zero-field splitting (ZFS) to the obtained distance information [7].…”

Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR

“…Recently, Gd(III) spin labels have attracted attention for in-cell PELDOR because they are resistant to the reducing environment inside the cell. 18,19 The use of PELDOR in-cell has been demonstrated using spin-labeled ubiquitin, which was introduced into oocytes or HeLa cells. 19,20 Distance measurements have been reported on spin labeled colicin A added to E. coli .…”