The interactions of biological macromolecules with solvating water are fundamental to their structure, dynamics, and function1
. Historically, comprehensive site-resolved experimental insight into the behavior of solvent near protein surfaces has been notoriously difficult to obtain in solution. Some years ago, Wüthrich and coworkers employed multi-dimensional solution NMR to observe site-resolved dipolar magnetization exchange between protein and water molecules having relatively long-lived interactions2
and have provided insight into various properties of water intimately involved in the structure of proteins3
. In principle, the same NMR-based methodology could also allow access to a comprehensive site-resolved characterization of the interaction of water across the surface of a protein. Unfortunately this approach has been confounded by the extremely short residence times of hydration water, by complications arising from hydrogen exchange phenomena and by a potential ambiguity in the distinction between long-range contributions from water molecules in the bulk from those of the hydration waters of interest.
It is now well established that the motion of water molecules is somewhat slowed as a result of interaction with the protein surface4
. A variety of methods have shown that the 1–2 layers of water molecules nearest the macromolecular surface are motionally slowed though estimates vary between several-fold to perhaps two orders of magnitude5–8
. Regardless, hydration water-protein interactions generally remain sufficiently short-lived to quench the intermolecular dipolar magnetization exchange necessary to detect them using the nuclear Overhauser effect in the laboratory (NOE) or the rotating frame (ROE)9
. The situation is also considerably clouded by the inherent presence of exchange of hydrogens of the solvent with labile sites on the protein2,3,9
. Furthermore, a more recent theoretical analysis also predicts dominant contributions from long-range intermolecular dipolar couplings10
. This effect arises primarily from two geometric sources that combine to reduce the effective distance dependence of the NOE and ROE from the familiar inverse sixth-power dependence to a more slowly varying simple inverse dependence10–12
. These restrictions and complications have largely frustrated the exploration of the protein-water interface using high-resolution NMR methods5,13
. Here we take advantage of several favorable aspects of reverse micelle encapsulation to circumvent these limitations, enabling site-resolved measurement of the hydration water dynamics for human ubiquitin.