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Description of Hydration Water in Protein (GFP) Solution Stefania Perticaroli, Georg Ehlers, Christopher B. Stanley, Eugene Mamontov, Hugh M. O'Neill, Qiu Zhang, Xiaolin Cheng, Dean A. A. Myles, John Katsaras, and Jonathan D. Nickels J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b08845 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on October 28, 2016
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Journal of the American Chemical Society
Description of Hydration Water in Protein (GFP) Solution Stefania Perticaroli1,3‡*, Georg Ehlers2, Christopher B. Stanley3, Eugene Mamontov4, Hugh O’Neill3,6, Qiu Zhang3, Xiaolin Cheng5,6, Dean A. A. Myles3, John Katsaras1,3,7, Jonathan D. Nickels1,3,7‡* 1
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Shull Wollan Center, a Joint Institute for Neutron Sciences, Quantum Condensed Matter Division, Biology and 4 5 Soft Matter Division, Chemical and Engineering Materials Division, Center for Molecular Biophysics, Oak Ridge 6 National Laboratory, Oak Ridge, Tennessee 37831. Department of Biochemistry and Cellular and Molecular Biology, 7 Department of Physics and Astronomy, University of Tennessee, Knoxville, Tennessee 37996, United States KEYWORDS: Structure, dynamics, perturbation, SANS, neutron spectroscopy, crowding.
ABSTRACT: The structurally and dynamically perturbed hydration shells that surround proteins and biomolecules have a substantial influence upon their function and stability. This makes the extent and degree of water perturbation of practical interest for general biological study and industrial formulation. We present an experimental description of the dynamical perturbation of hydration water around green fluorescent protein in solution. Less than two shells (~5.5 Å) were perturbed, with dynamics a factor of 2‒10 times slower than bulk water, depending on their distance from the protein surface and the probe length of the measurement. This dependence on probe length demonstrates that hydration water undergoes sub-diffusive motions ( ∝ . for the first hydration shell, ∝ . for perturbed waters in the second shell), an important difference with neat water which demonstrates diffusive behavior ( ∝ ). These results help clarify the seemingly conflicting range of values reported for hydration water retardation as a logical consequence of the different length scales probed by the analytical techniques used.
INTRODUCTION The term, hydration water, describes the structurally and dynamically perturbed water surrounding proteins and biomolecules. This population of water has a defining influence on the structure and function of biomolecules1,2, especially proteins3; implying a fundamental connection between the dynamical properties of hydration water and many vital biochemical processes including protein folding, protein–ligand recognition, membrane, enzyme function, and DNA stability4-8. In the food, cosmetics, and pharmaceutical industries6,9, hydration water impacts properties such as solubility, emulsification, foamability, viscosity, gelation and degradation9. Despite this obvious importance, a robust description of the hydration population of water has been challenging. Certainly, part of this derives from the heterogeneity of the hydration shell at a molecular scale. Topological disorder of the biomolecule surface and the chemical heterogeneity of the solventexposed groups result in a diverse molecular environment, with hydration water exhibiting different dynamics10-24 relative to neat water. The way that these effects manifest in various experimental techniques can lead to discrepancies based on the time and length scales inherent in the measurements and property which is probed25. Two parameters are typically used to describe the extent and magnitude of this perturbation; the hydration
number, NH, and the retardation factor, ξ. NH describes the extent of the perturbation in terms of the number of water molecules surrounding the protein, whose physical properties are altered, as defined by a specific technique or analysis. ξ quantifies the magnitude of the perturbation as the ratio of characteristic relaxation times for hydration water relative to bulk, τHYDR/τBULK. This has the advantage of being less dependent on the specific correlation functions analyzed than reporting the absolute values of τ obtained from a specific technique. However, there are discrepancies in the literature about the magnitude of ξ, with results from NMR12,22 and MD simulations13,23,26 suggesting a slowdown of 2-5 times in the first hydration shell, while time-resolved fluorescence spectroscopy5,17,27 reports a significant proportion of the water population being slowed by one to two orders of magnitude. Neutron scattering (NS) spectroscopy is a twodimensional technique that provides information about atomic motions in both time and space, with accessible length scales from Ångstroms to nanometers; with broad dynamic ranges from hundreds of nanoseconds to femtoseconds that can be accessed by combining spectra from more than one instrument. Moreover, due to the large incoherent scattering cross section of hydrogen (H), larger than its isotope deuterium (D) or other elements (C, O,
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Figure 1. Small angle neutron scattering of GFP solutions. GFP solutions were prepared in D2O and measured at 283K. A) Solution scattering from GFP at 5 mg/ml was found to be well modeled using an elliptical cylinder. An estimate of the molecular weight was made from the forward scattering (29.8 kDa versus 27 kDa predicted from sequence). B) Protein-protein correlations in the 100 mg/ml solution result in a structure factor typical of repulsive particles. C) Due to the high aspect ratio of GFP, a hard sphere approximation does not accurately reproduce this structure factor. Therefore, an approximation is made taking qmax to be the nearest neighbor distance and the solution structure equated to a close packed FCC lattice, with GFP at the atom positions. This treatment models the data well and returns a volume fraction of 8.2% for GFP at 100 mg/ml in solution (equivalent to ~98 mg/ml). (One chain from the 1GFL structure was used in creating these figures)
N, S), NS provides the notable opportunity to distinguish the dynamics of the solvent from those of the protein, or vice-versa, through isotopic sensitivity. This makes NS ideal to study water diffusion, rotation, collective motions, and other interatomic interactions28. Importantly, the characteristic time scale of the probed single molecule translational-rotational dynamics is fast in comparison to the residence time of the water molecules within the shell. This means that the slow exchange condition29 is met and the two populations of water (hydration and bulk) can be distinguished. In this way, NS experiments provide simultaneous estimates of NH and ξ at a range of experimental probe lengths, revealing the underlying relationship of probe distance and observed ξ. In this work, we study hydration water in a 100 mg/ml in solution of green fluorescent protein (GFP). This concentration was chosen for its biological and biotechnological relevance. Total concentrations of up to 400 mg/ml have been reported in cellular compartments, perturbing water motions30,31. Similarly high protein concentrations, up to, or greater than 100 mg/ml are also found in therapeutic formulations, such as for monoclonal antibodies, due to high dosage (∼100 mg) therapies and the volume limit (
