Probing Protein Conformation in Cells by EPR Distance Measurements using Gd3+ Spin Labeling
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- 作者：Andrea Martorana ; Giuliano Bellapadrona ; Akiva Feintuch ; Enza Di Gregorio ; Silvio Aime ; Daniella Goldfarb
- 刊名：Journal of the American Chemical Society
- 出版年：2014
- 出版时间：September 24, 2014
- 年：2014
- 卷：136
- 期：38
- 页码：13458-13465
- 全文大小：433K
- ISSN：1520-5126
文摘
Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electron鈥揺lectron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd3+ chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells.