Nucleotide Spin Labeling for ESR Spectroscopy of ATP-Binding Proteins
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Site-directed spin labeling of proteins by chemical modification of engineered cysteine residues with the molecule MTSSL (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate) has been an invaluable tool for conducting double electron electron resonance (DEER) spectroscopy experiments. However, this method is generally limited to recombinant proteins with a limited number of reactive Cys residues that when modified will not impair protein function. Here, we present a method that allows for spin labeling of protein-nucleotide-binding sites by adenosine diphosphate (ADP) modified with a nitroxide moiety on the β-phosphate (ADP-β-S-SL). The synthesis of this ADP analog is straightforward and isolation of pure product is readily achieved on a standard reverse-phase high-performance liquid chromatography (HPLC) system. Furthermore, analyses of isolated ADP-β-S-SL by LC–mass spectrometry confirm that the molecule is very stable under ambient conditions. The crystal structure of ADP-β-S-SL bound to the ATP pocket of the histidine kinase CheA reveals specific targeting of the probe, whose nitroxide moiety is mobile on the protein surface. Continuous wave and pulsed-ESR measurements demonstrate the capability of ADP-β-S-SL to report on active site environment and provide reliable DEER distance constraints.
This work supported by a National Science Foundation (NSF) Graduate Research Fellowship to ARM (2014155468) and a National Institutes of Health grant to BRC (R35GM122535). We are grateful to the National Biomedical Center for Advanced ESR Technology supported through NIH grant P41GM103521 to J.R. Freed and NE-CAT of the Advanced Photon Source at Argonne National Labs (NIH Grant P41GM103403) for access to X-ray data collection facilities. Coordinates were deposited in the Protein Data Bank under ID 6MI6. We thank Siddharth Chandrasekaran and Daniyal Tariq for assistance with cw-ESR experiments, Madhur Srivastava for help with data processing and Peter Borbat for helpful discussions.
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