Protein Hydration Dynamics in Aqueous Solution: A Comparison of Bovine Pancreatic Trypsin Inhibitor and Ubiquitin by Oxygen-17 Spin Relaxation Dispersion

Abstract

Water oxygen-17 spin relaxation was used to study hydration and dynamics Condensed Matter Magnetic Resonance Group, Chemical of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin in aqueous solution. The frequency dispersion of the longitudinal Center, Lund University P. O. Box 124, S-22100 Lund and transverse relaxation rates was measured over the Larmor frequency Sweden range 2.6 to 49 MHz in the pD range 2 to 11 at 27°C. While the protein-induced relaxation enhancement was similar for the two proteins at high frequencies, it was an order of magnitude smaller for ubiquitin than for BPTI at low frequencies. This difference was ascribed to the abscence, in ubiquitin, of highly ordered internal water molecules, which are known to be present in BPTI and in most other globular proteins. These observations demonstrate that the water relaxation dispersion in protein solutions is essentially due to a few structural water molecules buried within the protein matrix, but exchanging rapidly with the external water. The relaxation data indicate that the internal water molecules of BPTI exchange with bulk water on the time-scale 10 −8 to 10 −6 second thus lowering the recently reported upper bound on the residence time of these internal water molecules by four orders of magnitude, and implying that local unfolding occurs on the submicrosecond time-scale. The water molecules residing at the surface of the two proteins were found to be highly mobile, with an average rotational correlation time of approximately 20 picoseconds. For both proteins, the oxygen-17 relaxation depended only very weakly on pD, showing that ionic residues do not perturb hydration water dynamics more than other surface residues. We believe that the present results resolve the long-standing controversy regarding the mechanism behind the spin relaxation dispersion of water nuclei in protein solutions, thus establishing oxygen-17 relaxation as a powerful tool for studies of structurally and functionally important water molecules in proteins and other biomolecules.

Biomolecular hydration: From water dynamics to hydrodynamics

“…Although important for small ions, this electrohydrodynamic effect can be shown to be negligible for proteins. This theoretical prediction is borne out by MRD experiments on the proteins BPTI and ubiquitin (54,55), showing that D R is nearly constant in the pH range 2-11. Moreover, the modest variation (20-30%) seen in D R does not correlate with either the net protein charge or the total number of charged groups.…”

Simulations of the confinement of ubiquitin in self-assembled reverse micelles

“…Pocket-U and pocket-V are named due to the shape of pocket region. The hydration of Pocket-V has been studied by Denisov et al, 59,60 and water in this pocket is responsible for the 17 O relaxation dispersion. The lifetime for a long-lived buried water molecule in ubiquitin has been estimated to be 60-90 ns.…”

Protein surface hydration mapped by site-specific mutations

“…The long nanosecond dynamics represents the wholeprotein tumbling motion. The early small decays, on the time scales from 62 to 464 ps, result from the local wobbling motions with semiangles of [11][12][13][14][15][16][17][18][19] o (30). The time scales of 62-464 ps do not correlate with those of solvation dynamics (Fig.…”

“…A theoretical model was developed to take into account the exchange with bulk water (4,12), and the dynamics are consistent with molecular dynamics (MD) simulations of residence times (13)(14)(15)(16) on time scales from femtoseconds to picoseconds. Earlier NMR studies have reported hydration dynamics (residence times) in the subnanosecond regime (17)(18)(19)(20), but, more recently, a claim has been made that water motions at protein surfaces are ultrafast compared with bulk water, only slowing down by a factor of two to three (21,22). This Ͻ10-ps range would imply that the observed long-time hydration dynamics in tens of picoseconds are due to protein side-chain relaxation (22,23).…”