Abstract
In-cell EPR in combination with site-directed spin-labeling is a very powerful tool to monitor the structure and dynamics of bio-macromolecules in their natural environment. We have demonstrated that it can be used for precise intracellular distance measurements as proven by a model compound, which consists of a spacer of well-known stiffness. Additionally, as model peptide an oligoproline peptide was site-directedly spin labeled and analyzed by in-cell EPR. The results suggest that the peptide is inserted into cell membranes, coinciding with a conformational change. We investigated the in-cell conformations of the Parkinson-Protein alpha-Synuclein and its disease variants. The most elegant approach for site-directed spin labeling is the genetic encoding of a noncanonical, spin-labeled amino acid. This enables the intracellular biosynthesis of spin-labeled proteins and obviates the need for any chemical labeling step usually required for protein EPR studies. We have developed a genetically encoded spin label that can be introduced at multiple, user-defined sites of a protein. It can report intramolecular distance distributions in proteins by EPR measurements. This provides elegant new perspectives for in-cell EPR studies of endogenous proteins.
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