1 research outputs found
Gd(III)-PyMTA Label Is Suitable for In-Cell EPR
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 GdÂ(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 <i>Xenopus laevis</i> oocytes at 18 °C for more than 24 h. The proline rich peptide
H-AP<sub>10</sub>CP<sub>10</sub>CP<sub>10</sub>-NH<sub>2</sub> was
site-directedly spin labeled with Gd-PyMTA at both cysteine moieties.
The resulting peptide, H-AP<sub>10</sub>CÂ(Gd-PyMTA)ÂP<sub>10</sub>CÂ(Gd-PyMTA)ÂP<sub>10</sub>-NH<sub>2</sub>, as well as the model compound Gd-spacer-Gd,
which consists of a spacer of well-known stiffness, were microinjected
into <i>Xenopus laevis</i> oocytes, and the GdÂ(III)–GdÂ(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 GdÂ(III) ion and the C<sub>α</sub> of
the cysteine moiety into account. The results suggest that the spin
labeled peptide H-AP<sub>10</sub>CÂ(Gd-PyMTA)ÂP<sub>10</sub>CÂ(Gd-PyMTA)ÂP<sub>10</sub>-NH<sub>2</sub> is inserted into cell membranes, coinciding
with a conformational change of the oligoproline from a PPII into
a PPI helix