EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels
Citations Over TimeTop 10% of 2015 papers
Abstract
The genetic encoding of nitroxide amino acids in combination with electron paramagnetic resonance (EPR) distance measurements enables precise structural studies of native proteins, i.e. without the need for mutations to create unique reactive sites for chemical labeling and thus with minimal structural perturbation. We here report on in vitro DEER measurements in native E. coli thioredoxin (TRX) that establish the nitroxide amino acid SLK-1 as a spectroscopic probe that reports distances and conformational flexibilities in the enzyme with nonmutated catalytic centers that are not accessible by the use of the traditional methanethiosulfonate spin label (MTSSL). We generated a rotamer library for SLK-1 that in combination with molecular dynamics (MD) simulation enables predictions of distance distributions between two SLK-1 labels incorporated into a target protein. Toward a routine use of SLK-1 for EPR distance measurements in proteins and the advancement of the approach to intracellular environments, we study the stability of SLK-1 in E. coli cultures and lysates and establish guidelines for protein expression and purification that offer maximal nitroxide stability. These advancements and insights provide new perspectives for facile structural studies of native, endogenous proteins by EPR distance measurements.
Related Papers
- → Structural Origins of Nitroxide Side Chain Dynamics on Membrane Protein α-Helical Sites,(2010)64 cited
- → Dap‐SL: a new site‐directed nitroxide spin labeling approach for determining structure and motions in synthesized peptides and proteins(2002)24 cited
- → Diversification of EPR signatures in site directed spin labeling using a β-phosphorylated nitroxide(2014)16 cited
- → Nitroxide Spin Label Side Chain Dynamics of Solvent Exposed Sites on Membrane Proteins(2011)
- → Analysis of the Electron Spin Resonance of Spin Labels Using Chemometric Methods(1977)