Tracing protein native radicals under in vitro and in vivo conditions via EPR

Abstract

Although more than a hundred thousand protein structures are registered in the Protein Data Bank, there are still more open questions than answers. One of these proteins that have captivated researchers for decades is ribonucleotide reductase, short RNR. Due to its central role in every living organism, RNR has been the focus of research on several occasions, but many questions remain unanswered despite numerous studies. Moreover, information about RNR structure in its natural environment, namely living cells, is entirely lacking. A method that can be used for protein structure elucidation is electron paramagnetic resonance, shortly EPR. EPR is also widely used for RNR studies since the enzyme has a native radical that is used for catalysis. In this work, state-of-the-art EPR spectroscopy was used to address some of the open questions related to RNR. Advanced EPR techniques were used to determine the structure of the stable tyrosyl radical Y122• in E. coli class Ia RNR in living whole E. coli cells at high resolution. Furthermore, EPR was used to investigate the in vivo radical distribution of RNR. In addition to in-cell EPR, three other applications of magnetic resonance spectroscopy are highlighted in this work. First, insights into the development process of a new spin label are given, using RNR as a model system. Through these studies, azidophenylalanine was identified as a suitable candidate that could contribute to the field of in-cell protein structure elucidation via spin labeling. Second, a high-pressure apparatus was used to study the effect of pressure on E. coli RNRs structure by EPR. Finally, independently of RNR, cw-EPR was used to characterize the long-lived C60 radical anion generated in a molecular coordination cage.