Affiliation:
1. Department of Physiology and Biophysics, University of Washington, Seattle, United States
Abstract
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.
Funder
National Eye Institute
National Institute of General Medical Sciences
National Institutes of Health
National Institute of Diabetes and Digestive and Kidney Diseases
National Institute of Mental Health
Publisher
eLife Sciences Publications, Ltd
Subject
General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience
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46 articles.
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