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Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.
Pubmed ID: 29923827 RIS Download
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View all literature mentionsCell line HEK293T/17 is a Transformed cell line with a species of origin Homo sapiens (Human)
View all literature mentionsCell line HEK293T/17 is a Transformed cell line with a species of origin Homo sapiens (Human)
View all literature mentionsCell line HEK293T/17 is a Transformed cell line with a species of origin Homo sapiens (Human)
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