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Watching Single Unmodified Enzymes at Work

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posted on 2023-06-08, 12:46 authored by Cuifeng Ying, Edona Karakaci, Esteban Bermudez-Urena, Alessandro Ianiro, Ceri Foster, Saurabh Awasthi, Anirvan Guha, Louise Bryan, Jonathan List, Sandor Balog, Guillermo P. Acuna, Reuven Gordon, Michael Mayer
Many proteins undergo conformational changes during their activity. A full understanding of the function of these proteins can only be obtained if different conformations and transitions between them can be monitored in aqueous solution, with adequate temporal resolution and, ideally, on a single-molecule level. Interrogating conformational dynamics of single proteins remains, however, exquisitely challenging and typically requires site-directed chemical modification combined with rigorous minimization of possible artifacts. These obstacles limit the number of single-protein investigations. The work presented here introduces an approach that traps single unmodified proteins from solution in a plasmonic hotspot and makes it possible to assign changes in refractive index to changes in protein conformation while monitoring these changes for minutes to hours with a temporal resolution at least as fast as 40 microseconds. The resulting single molecule data reveals that adenylate kinase employs a hidden enzymatic sub-cycle during catalysis, that citrate synthase populates a previously unknown intermediate conformation, which is more important for its enzymatic activity than its well-known open conformation, that hemoglobin transitions in several steps from its deoxygenated and rigid T state to its oxygenated and flexible R state, and that apo-calmodulin thermally unfolds and refolds in steps that correspond to conformational changes of individual protein domains.



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