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A Reactive, Rigid GdIII Labeling Tag for In-Cell EPR Distance Measurements in Proteins.
- Source :
-
Angewandte Chemie International Edition . 3/6/2017, Vol. 56 Issue 11, p2914-2918. 5p. - Publication Year :
- 2017
-
Abstract
- 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 GdIII 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-GdIII 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 μ m. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 14337851
- Volume :
- 56
- Issue :
- 11
- Database :
- Academic Search Index
- Journal :
- Angewandte Chemie International Edition
- Publication Type :
- Academic Journal
- Accession number :
- 121574376
- Full Text :
- https://doi.org/10.1002/anie.201611051