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

Authors
Ching, H. Y. Vincent; Mascali, Florencia Carla; Bertrand, Hélène C.; Bruch, Eduardo Marcos; Demay Drouhard, Paul; Rasia, Rodolfo Maximiliano; Policar, Clotilde; Tabares, Leandro Cesar; Un, Sun
Publication Year
2016
Language
English
Format
article
Status
Published version
Description
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.
Fil: Ching, H. Y. Vincent. Université Paris-Saclay; Francia
Fil: Mascali, Florencia Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Bertrand, Hélène C.. Ecole Normale Supérieure; Francia. Centre National de la Recherche Scientifique; Francia. Universite de Paris VI; Francia
Fil: Bruch, Eduardo Marcos. Université Paris-Saclay; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Demay Drouhard, Paul. Ecole Normale Supérieure; Francia. Centre National de la Recherche Scientifique; Francia. Universite de Paris VI; Francia
Fil: Rasia, Rodolfo Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Policar, Clotilde. Ecole Normale Supérieure; Francia. Centre National de la Recherche Scientifique; Francia. Universite de Paris VI; Francia
Fil: Tabares, Leandro Cesar. Université Paris-Saclay; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Un, Sun. Université Paris-Saclay; Francia
Subject
MN(II)
EPR
DEER
Otras Ciencias Físicas
Ciencias Físicas
CIENCIAS NATURALES Y EXACTAS
Otras Ciencias Biológicas
Ciencias Biológicas
CIENCIAS NATURALES Y EXACTAS
Access level
Restricted access
License
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repository
CONICET Digital (CONICET)
Institution
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identifier
oai:ri.conicet.gov.ar:11336/61004