The Use of Mn(II) Bound to His-tags as Genetically Encodable Spin-Label for Nanometric Distance Determination in Proteins.
J Phys Chem Lett
; 7(6): 1072-6, 2016 Mar 17.
Article
en En
| MEDLINE
| ID: mdl-26938795
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Marcadores de Spin
/
Proteínas
/
Histidina
/
Manganeso
Idioma:
En
Revista:
J Phys Chem Lett
Año:
2016
Tipo del documento:
Article
País de afiliación:
Francia