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. 2014 Jan 8;136(1):188-94.
doi: 10.1021/ja407858c. Epub 2013 Dec 16.

Crowding induced collective hydration of biological macromolecules over extended distances

John T King  1 , Evan J Arthur, Charles L Brooks 3rd, Kevin J Kubarych
Affiliations

Crowding induced collective hydration of biological macromolecules over extended distances

John T King et al. J Am Chem Soc. .

Abstract

Ultrafast two-dimensional infrared (2D-IR) spectroscopy reveals picosecond protein and hydration dynamics of crowded hen egg white lysozyme (HEWL) labeled with a metal-carbonyl vibrational probe covalently attached to a solvent accessible His residue. HEWL is systematically crowded alternatively with polyethylene glycol (PEG) or excess lysozyme in order to distinguish the chemically inert polymer from the complex electrostatic profile of the protein crowder. The results are threefold: (1) A sharp dynamical jamming-like transition is observed in the picosecond protein and hydration dynamics that is attributed to an independent-to-collective hydration transition induced by macromolecular crowding that slows the hydration dynamics up to an order of magnitude relative to bulk water. (2) The interprotein distance at which the transition occurs suggests collective hydration of proteins over distances of 30-40 Å. (3) Comparing the crowding effects of PEG400 to our previously reported experiments using glycerol exposes fundamental differences between small and macromolecular crowding agents.

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Figures

Fig 1
Fig 1
Crystal structure of HEWL-RC, linear and 2D-IR spectra, example FFCF. a, Structure of the metal-carbonyl vibrational probe and the crystal structure of the His 15 labeled HEWL carbonyl complex (probe site highlighted in yellow). b, Linear FTIR spectrum and (c) 2DIR spectrum shown for the metal-carbonyl CO region. d, Example of a typical frequency-frequency correlation function, showing an initial decay on the order of a few picoseconds corresponding to the hydration dynamics, followed by a static offset due to protein inhomogeneity that is not sampled within the experimental window.
Fig. 2
Fig. 2
Interfacial water and protein dynamics of HEWL-RC in D2O/glycerol mixtures. a, FFCFs for HEWL-RC in D2O/PEG mixtures, ranging from pure D2O to 80% PEG by volume. b, Hydration timescale, obtained by the initial decay of the correlation function, and the protein dynamics, estimated by the static offset of the correlation function, plotted as a function of solvent composition. A strong coupling is clear from the data, with both the hydration and protein dynamics slowing down as glycerol is added to the system. There is also a sharp dynamic transition occurring at roughly 60% PEG. We suggest this transition results from the extended protein hydration environment overlapping with the PEG hydration environment. c, The vibrational relaxation—estimated from the rephasing signal amplitude—lacks any PEG400 dependence suggesting that the protein remains fully hydrated in the region around the probe.
Fig. 3
Fig. 3
Comparison of interfacial water dynamics of HEWL-RC in solutions of glycerol and PEG400. While the magnitude of the hydration dynamics slowdown induced by each cosolvent is similar at high concentrations, the dynamic transition is observed only in the presence of the macromolecular crowding agent.
Fig. 4
Fig. 4
Interfacial water and protein dynamics of HEWL-RC in the presence of excess lysozyme. a, FFCFs for HEWL-RC in self-crowding conditions, ranging from 20 mg/mL to 160 mg/mL. b, Hydration timescale, obtained by the initial decay of the correlation function, and the protein dynamics, estimated by the static offset of the correlation function, plotted as a function of solvent composition. A strong coupling is clear from the data, with both the hydration and protein dynamics slowing down as excess lysozyme is added to the system. Similar to the PEG400 crowding, a dynamical transition is observed at sufficient crowding, though this transition occurs at lower concentrations of HEWL because of the more significant constraining effect that HEWL has on surrounding waters. c, Vibrational lifetimes estimated through the signal amplitude of the rephasing spectrum again show a consistently short lifetime, consistent with a lack of protein-protein interactions that would result in surface dehydration and increased lifetimes.
Fig. 5
Fig. 5
Hydration and protein dynamics of HEWL-RC in crowding conditions plotted as a function of protein-protein distance. a, The protein-protein distance is defined as the average surface-to-surface distance between proteins using a spherical approximation, which can be estimated for each concentration. b, Assuming a homogeneous mixture, the average surface-to-surface distance between proteins can be estimated, revealing that the transition occurs at a protein-protein distance of 30-40 Å.
Fig. 6
Fig. 6
Example of the simulation analysis where (a) two proteins are separated by a set distance d and the bridging water is selected for analysis and (b) four proteins are arranged tetrahedrally, all of which are separated by the same variable distance. The water that was selected for analysis is shown. c, Hydrogen bond number of the crowded water as a function of protein-protein distance. In each case there is no clear transition in the average hydrogen bonds per water molecule, suggesting no significant change in structure. A slight downward trend is observed as the interprotein distance is reduced, though this is the result of a higher relative contribution from the interfacial water, which has fewer hydrogen bonds than bulk water. d, Hydrogen bond correlation times of the crowded water as a function of protein-protein distance. The occurrence of a dynamic transition is found between 10-15 Å for two proteins and 20-25 Å for the four protein simulation. In each case, only a weak coupling is observed before and after the dynamic transition. The results not only demonstrate a percolation-like transition of water dynamics upon crowding, but also show that the distance of this transition is a function of the degree and geometry of crowding.
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