Protein-water interaction studied by solvent 1H, 2H, and 17O magnetic relaxation.

Abstract

Previous studies of the magnetic field de- Solvent proton relaxation in protein solutions can be accounted for reasonably well within the framework of a simple model of water exchange between the bulk solvent, in which the protons are assumed not to interact with the solute, and regions on or near the protein where the rotational motion of the protein molecules is conveyed to the water molecules. If it is assumed that the water molecules in the latter region attach rigidly to the protein, then one can readily estimate (1) the number n of such attachment sites to be about 3 for lysozyme and to increase at least as fast as the surface area for larger proteins (K. Hallenga and S. H. Koenig, to be published). If the attachment is assumed not rigid, the derived value of n is greater by a small factor, but is still-and this is the major unsettling feature of this simple two-site model-only a few percent of the number of water molecules in the first hydration layer (1). The nature of such special sites is difficult to conceive.From the data for proton relaxation, one can only set fairly wide limits on the value (or distribution of values) of TM, the mean lifetime of a water molecule on the protein and a quantity which is a feature of the model itself. The application of the model gives TM > TR 10-7 sec, and TM < Tipr -10-4 sec, the relaxation time of the protons when on the protein. In an attempt to get a better measurement of TM, as well as to test other predictions of the models, we measured and report here comparisons of the magnetic field dependence of the relaxation rate of 170 in solutions of protein in 17O-enriched water, and deuterons in 2H20 solutions, with the proton data. Since deuterons in water relax about 10 times faster than protons, and 170 nuclei about 100 times faster than deuterons, the upper limit on the model value for TM is drastically reduced and the range for TM is drastically narrowed. We have also made measurements on an extremely large protein-hemocyanin associated to a M.W. of 9 X 106-in order to increase the lower limit of TrM. The experiments were quite successful in that -TM was squeezed into nonexistence: i.e., the data reported below cannot be explained by a simple two-region model with a defined life-

Proton and Deuteron Relaxation Study of Molecular Dynamics in Lysozyme Solutions

“…The size of the magnetization components obtained from the decompositions was independent of frequency and similar to the size of the corresponding fraction determined in the 2DTE experiment at 40 MHz (Table I). In part, the proton 1/T1 dispersion of the Li-component in N-HEWL and the deuteron 1/T1 dispersion of the Li-component in D-HEWL have been reported in the literature [21,33]. The measurements in the literature extend to lower frequencies, whereas our measurements include data at higher frequencies.…”

“…In addition, exchange of labile protein hydrogens may play an important role [29]. However, it has been shown [33] that in lysozyme solution, at pH 7, labile protein hydrogens do not make any measurable contribution to solvent proton relaxation rates, and this effect is assumed negligible in the present study.…”

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

“…This removes the internal inconsistencies that led some authors (Koenig et al, 1975;Bryant, 1988;Koenig & Brown, 1991) to reject a simple fast-exchange model. Furthermore, the hitherto controversial mechanism whereby the protein rotational motion is conveyed to the water molecules (Bryant, 1988) is now identified as the exchange, on the time-scale 10 −8 to 10 −6 second (for 17 O relaxation and small proteins), of highly ordered internal water molecules.…”

Novel Insights Into Protein Structure and Dynamics Utilizing the Red Edge Excitation Shift Approach

“…12 It is estimated that a threshold level of hydration (less than 0.4 grams of water per gram of protein) is required to fully activate the dynamics and function of globular proteins. [13][14][15] The most direct evidence for the importance of water in protein structure and function is that in the absence of water, proteins cannot diffuse and become non-functional. Lack of motion and function have been observed when proteins are transferred to organic solvents 16 and dehydration studies show that at least a monolayer of water molecules is required for the protein to be fully functional.…”