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

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• 作者：Mian Qi ; Andreas Gro脽 ; Gunnar Jeschke ; Adelheid Godt ; Malte Drescher
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：October 29, 2014
• 年：2014
• 卷：136
• 期：43
• 页码：15366-15378
• 全文大小：636K
• ISSN：1520-5126

文摘

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 掳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)鈥揋d(III) distances were determined by double electron鈥揺lectron 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_伪 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.