Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 56, Issue 11, Pages 2914-2918Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201611051
Keywords
EPR spectroscopy; protein dynamics; protein structures; spin labels; structure elucidation
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Funding
- Major National Scientific Research Projects [2013CB910201, 2016YFA0501202]
- National Natural Science Foundation of China [21473095, 21273121]
- Israel Science Foundation (ISF) [334/14]
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The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in-cell distance measurements by the double electron-electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd-III spin label, a thiol-specific DOTA-derivative (DO3MA-3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in-cell distance measurements by the DEER method carried out at W-band frequencies. The high performance of DO3MA-3BrPy-Gd-III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High-quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in-cell protein concentrations as low as 5-10 mu M.
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