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

摘要: 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 protein by electron resonance (EPR). Here, we demonstrate pulse electron–electron double (DEER) measurements on spin-labels consisting Mn(II) ions coordinated to sequence histidines, so-called His-tags, that are ubiquitously added genetic engineering facilitate purification. Although affinity His-tags was low (800 μM), Mn(II)-bound yielded readily detectable DEER time traces even at concentrations expected in cells. We were able determine accurately distance between two His-tag ends rigid helical polyproline peptide known structure, as well completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies labeling procedure but also represents first step towards usin...