1536470-16-2Relevant academic research and scientific papers
Genetically Encoded Spin Labels for in Vitro and In-Cell EPR Studies of Native Proteins
Schmidt,Fedoseev,Summerer,Drescher
, p. 483 - 502 (2015)
Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful approach to study the structure, dynamics, and interactions of proteins. The genetic encoding of the noncanonical amino acid spin-labeled lysine 1 (SLK-1) eliminates the need for any chemical labeling steps in SDSL-EPR studies and enables the investigation of native, endogenous proteins with minimal structural perturbation, and without the need to create unique reactive sites for chemical labeling. We report detailed experimental procedures for the efficient synthesis of SLK-1, the expression and purification of SLK-1-containing proteins under conditions that ensure maximal integrity of the nitroxide radical moiety, and procedures for intramolecular EPR distance measurements in proteins by double electron-electron resonance.
A genetically encoded spin label for electron paramagnetic resonance distance measurements
Schmidt, Moritz J.,Borbas, Julia,Drescher, Malte,Summerer, Daniel
supporting information, p. 1238 - 1241 (2014/02/14)
We report the genetic encoding of a noncanonical, spin-labeled amino acid in Escherichia coli. This enables the intracellular biosynthesis of spin-labeled proteins and obviates the need for any chemical labeling step usually required for protein electron paramagnetic resonance (EPR) studies. The amino acid can be introduced at multiple, user-defined sites of a protein and is stable in E. coli even for prolonged expression times. It can report intramolecular distance distributions in proteins by double-electron electron resonance measurements. Moreover, the signal of spin-labeled protein can be selectively detected in cells. This provides elegant new perspectives for in-cell EPR studies of endogenous proteins.
