Neutrons describe ectoine effects on water H-bonding and hydration around a soluble protein and a cell membrane

Abstract

Understanding adaptation to extreme environments remains a challenge of high biotechnological potential for fundamental molecular biology. The cytosol of many microorganisms, isolated from saline environments, reversibly accumulates molar concentrations of the osmolyte ectoine to counterbalance fluctuating external salt concentrations. Although they have been studied extensively by thermodynamic and spectroscopic methods, direct experimental structural data have, so far, been lacking on ectoine-water-protein interactions. In this paper, in vivo deuterium labeling, small angle neutron scattering, neutron membrane diffraction and inelastic scattering are combined with neutron liquids diffraction to characterize the extreme ectoine-containing solvent and its effects on purple membrane of H. salinarum and E. coli maltose binding protein. The data reveal that ectoine is excluded from the hydration layer at the membrane surface and does not affect membrane molecular dynamics, and prove a previous hypothesis that ectoine is excluded from a monolayer of dense hydration water around the soluble protein. Neutron liquids diffraction to atomic resolution shows how ectoine enhances the remarkable properties of H-bonds in water-properties that are essential for the proper organization, stabilization and dynamics of biological structures.

Figure 1. Stuhrmann plots H- and D-MBP in various solvents.

Square root of corrected forward scattered intensity (equal to particle excess scattering length ΔρV) versus solvent SLD for H-MBP and D-MBP in 2 M ectoine and 3 M ectoine H₂O and D₂O solvent.

Figure 2

(A) Schematic diagram of the particle made up of protein (red) surrounded by its hydration shell (light blue); the solvent is shown as dark blue. (B) Section showing the SLD distribution through solvent and H-MBP and D-MBP particles in 3 M ectoine in H₂O and D₂O. The particles are divided into their protein (red) and hydration shell of specific gravity 1.1 (blue) components. Solvent SLD levels are shown as straight lines at 0 and 5.456 × 10¹⁰ cm⁻².

Figure 3

(A) Lamellar diffraction from PM oriented membrane stacks under different conditions plotted against scattering angle (2θ in degrees). The main lamellar peaks correspond to a spacing of d ~ 82 Å. Note the significant increase in second order (indicated by red lines) intensity when H-ectoine is added to D₂O in the inter-lamellar hydration layer. The schematic diagram shows two membranes (purple) in the stack separated by a water layer (light blue); the green line in the top diagram indicates the position of ectoine. (B) In-plane diffraction for samples hydrated with H₂O, and (C) for samples hydrated with D₂O.

Figure 4

Logarithmic representation of the normalized intensities as a function of Q² at eight temperatures between 280 and 320 K for (A) H-PM/D₂O and (B) H-PM/D₂O/D-ectoine (b). The Q² range from which the MSD were extracted (0.18 Å⁻² < Q² < 1.33 Å⁻²), according to the Gaussian approximation (equation 9) is indicated by vertical lines. The straight lines in (A,B) indicate the linear fits at 320 K.

Figure 5. Mean square displacements as a function of temperature (from the data in Fig. 4) and calculated resilience values () on the nanosecond time scale for H-PM samples hydrated with D₂O in the presence and absence of D-ectoine (see text).

Figure 6

(A) Liquids diffraction of ectoine solutions. The radial distribution function g(r), obtained by Fourier transformation of the experimental structure factor S(Q) for sample 1.5 M D-ectoine/D₂O. The inter-molecular region, beyond r = 2 Å, is expanded in the inset, which also shows the H-bonding scheme between two adjacent D₂O molecules (see text). (B) Comparison between the structure factor of the D-labeled ectoine in D₂O (labeled DD) solution and the corresponding pure D₂O from Soper et al. (upper curve), and D-labeled ectoine in H₂O (labeled HH) and the corresponding pure H₂O (bottom curve). Significant differences between the ectoine solutions and pure water are apparent.

Figure 7. Plot of the ratio (ΔρV in 3 M ectoine H₂O)/(ΔρV in 3 M ectoine D₂O) vs X (V_Hydration/V_Protein) for D-MBP.

The red point with error bars indicates the experimental value of the ratio and corresponding X value. See Methods text.