title: The Use of Mn(II) Bound to His-tags as Genetically Encodable Spin-Label for Nanometric Distance Determination in Proteins.
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.
authors.affiliation: 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.
authors.forename: H Y Vincent
authors.initials: HY
authors.lastname: Ching
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario; Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario , 2000 Rosario, Argentina.
authors.forename: Florencia C
authors.initials: FC
authors.lastname: Mascali
authors.affiliation: Ecole Normale Supérieure-PSL Research University, Département de Chimie, Sorbonne Universités - UPMC Univ Paris 06, CNRS UMR 7203 LBM , F-75005 Paris, France.|CNRS, UMR 7203, Laboratoire des Biomolécules, F-75005 Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR 7203, Laboratoire des Biomolécules, F-75005 Paris, France.
authors.forename: Hélène C
authors.initials: HC
authors.lastname: Bertrand
authors.affiliation: 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.
authors.forename: Eduardo M
authors.initials: EM
authors.lastname: Bruch
authors.affiliation: Ecole Normale Supérieure-PSL Research University, Département de Chimie, Sorbonne Universités - UPMC Univ Paris 06, CNRS UMR 7203 LBM , F-75005 Paris, France.|CNRS, UMR 7203, Laboratoire des Biomolécules, F-75005 Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR 7203, Laboratoire des Biomolécules, F-75005 Paris, France.
authors.forename: Paul
authors.initials: P
authors.lastname: Demay-Drouhard
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario; Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario , 2000 Rosario, Argentina.
authors.forename: Rodolfo M
authors.initials: RM
authors.lastname: Rasia
authors.affiliation: Ecole Normale Supérieure-PSL Research University, Département de Chimie, Sorbonne Universités - UPMC Univ Paris 06, CNRS UMR 7203 LBM , F-75005 Paris, France.|CNRS, UMR 7203, Laboratoire des Biomolécules, F-75005 Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR 7203, Laboratoire des Biomolécules, F-75005 Paris, France.
authors.forename: Clotilde
authors.initials: C
authors.lastname: Policar
authors.affiliation: 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.
authors.forename: Leandro C
authors.initials: LC
authors.lastname: Tabares
authors.affiliation: 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.
authors.forename: Sun
authors.initials: S
authors.lastname: Un
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013113:Spin Labels
chemical_list: D010455:Peptides; D011506:Proteins; D013113:Spin Labels; D008345:Manganese; D006639:Histidine