  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.
