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Title: On the Use of Q-Band Double Electron–Electron Resonance To Resolve the Relative Orientations of Two Double Histidine-Bound Cu 2+ Ions in a Protein
Award ID(s):
1725678 1613007
NSF-PAR ID:
10098382
Author(s) / Creator(s):
; ; ; ;
Date Published:
Journal Name:
The Journal of Physical Chemistry B
Volume:
122
Issue:
47
ISSN:
1520-6106
Page Range / eLocation ID:
10669 to 10677
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Abstract

    Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins’ conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII19F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell GdIII19F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid GdIIItags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived GdIII19F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIIIand19F regions in the cell.

     
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  2. Abstract

    Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins’ conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII19F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell GdIII19F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid GdIIItags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived GdIII19F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIIIand19F regions in the cell.

     
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  3. null (Ed.)