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J Phys Chem Lett. 2016 Mar 17;7(6):1072-6. doi: 10.1021/acs.jpclett.6b00362. Epub 2016 Mar 8.

The Use of Mn(II) Bound to His-tags as Genetically Encodable Spin-Label for Nanometric Distance Determination in Proteins.

Author information

1
Institute for Integrative Biology of the Cell (I2BC), Department of Biochemistry, Biophysics and Structural Biology, Université Paris-Saclay, CEA, CNRS UMR 9198 , F-91191 Gif-sur-Yvette, France.
2
Instituto de Biología Molecular y Celular de Rosario; Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario , 2000 Rosario, Argentina.
3
Ecole Normale Supérieure-PSL Research University, Département de Chimie, Sorbonne Universités - UPMC Univ Paris 06, CNRS UMR 7203 LBM , F-75005 Paris, France.
4
CNRS, UMR 7203, Laboratoire des Biomolécules, F-75005 Paris, France.
5
Sorbonne Universités, UPMC Univ Paris 06, UMR 7203, Laboratoire des Biomolécules, F-75005 Paris, France.

Abstract

A genetically encodable paramagnetic spin-label capable of self-assembly from naturally available components would offer a means for studying the in-cell structure and interactions of a protein by electron paramagnetic resonance (EPR). Here, we demonstrate pulse electron-electron double resonance (DEER) measurements on spin-labels consisting of Mn(II) ions coordinated to a sequence of histidines, so-called His-tags, that are ubiquitously added by genetic engineering to facilitate protein purification. Although the affinity of His-tags for Mn(II) was low (800 μM), Mn(II)-bound His-tags yielded readily detectable DEER time traces even at concentrations expected in cells. We were able to determine accurately the distance between two His-tag Mn(II) spin-labels at the ends of a rigid helical polyproline peptide of known structure, as well as at the ends of a completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies the labeling procedure but also represents a first step towards using self-assembling metal spin-labels for in-cell distance measurements.

PMID:
26938795
DOI:
10.1021/acs.jpclett.6b00362
[Indexed for MEDLINE]

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