A Reactive, Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins. Issue 11 (1st February 2017)

• Record Type: Journal Article
• Authors: Yang, Yin; Yang, Feng; Gong, Yan‐Jun; Chen, Jia‐Liang; Goldfarb, Daniella; Su, Xun‐Cheng
• Is Part Of: Angewandte Chemie international edition. Volume 56:Issue 11(2017)
• Page Start: 2914
• Page End: 2918
• Publication Date: 2017-02-01
• Subjects: EPR spectroscopy -- protein dynamics -- protein structures -- spin labels -- structure elucidation; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201611051
• Languages: English
• ISSNs: 1433-7851

Abstract

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 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 μm . Abstract : Distance measurements : A reactive and rigid Gd III spin label showed excellent performances in distance measurements using the double electron–electron resonance (DEER) method in vitro and in cells. High‐quality DEER data could be obtained in HeLa cells at protein concentrations as low as 5–10 μm up to 12 h after the delivery of proteins.