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

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QI, Mian, Andreas GROSS, Gunnar JESCHKE, Adelheid GODT, Malte DRESCHER, 2014. Gd(III)-PyMTA Label Is Suitable for In-Cell EPR. In: Journal of the American Chemical Society. 136(43), pp. 15366-15378. ISSN 0002-7863. eISSN 1520-5126

@article{Qi2014Gd(II-30104, title={Gd(III)-PyMTA Label Is Suitable for In-Cell EPR}, year={2014}, doi={10.1021/ja508274d}, number={43}, volume={136}, issn={0002-7863}, journal={Journal of the American Chemical Society}, pages={15366--15378}, author={Qi, Mian and Groß, Andreas and Jeschke, Gunnar and Godt, Adelheid and Drescher, Malte} }

Groß, Andreas Gd(III)-PyMTA Label Is Suitable for In-Cell EPR Godt, Adelheid Jeschke, Gunnar Groß, Andreas Godt, Adelheid 2014 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-AP10CP<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>C(Gd-PyMTA)P<sub>10</sub>C(Gd-PyMTA)P<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 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 C<sub>α</sub> of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP<sub>10</sub>C(Gd-PyMTA)P<sub>10</sub>C(Gd-PyMTA)P<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. Jeschke, Gunnar 2015-02-27T10:34:03Z eng Qi, Mian Qi, Mian 2015-02-27T10:34:03Z Drescher, Malte Drescher, Malte

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