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Protection against Dehydration: a Neutron Diffraction Study on Aqueous Solutions of a Model Peptide and Trehalose. Michael Di Gioacchino, Fabio Bruni, and Maria Antonietta Ricci J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08046 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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The Journal of Physical Chemistry

Protection against Dehydration: a Neutron Diffraction Study on Aqueous Solutions of a Model Peptide and Trehalose. Michael Di Gioacchino, Fabio Bruni, and Maria Antonietta Ricci∗ Dipartimento di Scienze, Universit´a degli Studi Roma Tre, via della Vasca Navale 84, 00146-Roma, Italy E-mail: [email protected] Phone: +39 0655737226

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The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The ability of a wide class of organisms to reversibly go through cycles of suspended life and active metabolism, depending on the turnover of drought and normal water availability conditions, represents a challenging issue. The interest in the natural mechanism for drought survival has grown over time along with the request for always more efficient conservation techniques for biological materials. Carbohydrates, such as trehalose, accumulated in the cytoplasm of drought resistant cells, are considered responsible for desiccation tolerance. Nonetheless a detailed description of the interaction between trehalose and biomolecules is not yet established. Neutron diffraction experiments show that trehalose entraps a layer of water molecules in the first shell of a model peptide, N-Methylacetamide, without direct bonding with it. This evidence contrasts the hypothesis that trehalose substitutes water and supports the opposite view, namely of trehalose forming a protective shell which entraps a layer of water molecules at the surface of proteins, thus avoiding structural damage due to drought conditions.

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Introduction It is known since a long time 1–3 that there are relatively simple organisms such as plant seeds, spores, pollen grains, bacteria, nematodes, cysts of aquatic crustaceans, and even more complex ones such as the resurrection plant (Selaginella lepidophylla), that can survive to an almost complete removal of intracellular water, while their metabolism comes to a standstill. This state of suspended life (anhydrobiosis) can last as long as several years, or centuries and eventually, as soon as sufficient amount of water becomes available, the metabolic activity can turn back on. For many anhydrobiotic organisms going through cycles of drought and re-hydration is not only reversible, but also a physiological aspect of their life. The central question concerns the nature of the desiccation tolerance mechanism, peculiar of such organisms. While it has been observed that many drought tolerant organisms, before entering in a dry state, accumulate in the cytoplasm large amounts of carbohydrates 4–6 , there is no consensus about the specific function played by these compounds during desiccation and suspended life. As water is removed, the stresses imposed on membranes and other intracellular components may be very large 7 as shown by desiccation-intolerant organisms: in this case dehydration causes irreversible events, such as disruption of cellular and intracellular membranes, and denaturation of enzymes. Desiccation tolerance must be therefore achieved through a structural re-organization of the cytoplasmic components, possibly promoted by the presence of carbohydrates, e.g. trehalose. Indeed these are known to help stabilizing membranes and preserving structure and function of proteins in the absence of water, or during freezing 8–11 . Among simple and oligo-saccharides, trehalose is one of the most effective intracellular drought protectant 12–15 . To explain this observation, three hypothesis are available in the literature. Namely, either trehalose helps to vitrify the cytoplasm (vitrification scenario) 14,16,17 , thus blocking all metabolic processes requiring intracellular diffusion of metabolites, or it influences the structure of the hydration shell of metabolites. In this case, prevention of protein denaturation and damage of intracellular membranes upon dehydration can take place via 3



two possible processes. That is, either trehalose acts as a water substitute (water replacement scenario) 18 , or it entraps water at the surface of a protein or a membrane (water entrapment scenario) 19–21 . In all cases it preserves the molecular structure. Trehalose is not expressed in human cells, yet Levine and coworkers 22 have recently shown that engineered human cells containing trehalose can remain viable for days in the absence of water. This finding draws a renewed attention to the role of trehalose and its interaction with intracellular components, such as proteins. To shed light on this interaction, we have investigated by neutron diffraction the hydration of N-Methylacetamide (NMA) in the presence or absence of trehalose. NMA has been selected as a small molecule mimicking peptides, being composed of a single peptide bond 23 . The rationale behind our work is that the behavior of trehalose in our sample can be representative of its behavior in more complex biological media. We report evidence against the hypothesis that trehalose substitutes water and in favour of the segregation of a layer of water molecules in the first hydration shell of biomolecules.

Experimental technique and data handling Neutron diffraction with isotopic H/D substitution is an efficient method for studies of hydration of molecules and in particular of the hydrogen bond interaction in aqueous solutions, as neutrons are strongly scattered by hydrogen atoms and can distinguish the H and D isotopes 24 . The latter property allows recording richer information, compared to X-ray diffraction experiments, as shown below. Two experiments have been performed at the ISIS spallation source, on the SANDALS diffractometer 25 , by exploiting the isotopic H/D substitution 26 on both solvent (water) and solutes: The first experiment on a NMA/water solution with composition of 1 NMA molecule per 15 water molecules, the second one on a ternary NMA/trehalose/water solution, with composition of 1 NMA and 1 trehalose molecule per 100 water molecules. All experiments

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have been performed at ambient conditions.

Sample preparation All solutes have been purchased from Sigma-Aldrich. For the first experiment, N-Methylacetamide CH3 CONHCH3 (NMA) and its isotope CD3 CONDCD3 (NMA-7D) were dissolved either in H2 O or D2 O in the right proportions, in order to obtain the samples listed in Table 1. Table 1: Labels and composition of the investigated NMA-water samples. The suffix ”2-” identifies the binary solution. The NMA concentration is 3.71 M. Sample Label

NMA-7D (molar ratio) 2-deuterated 1 2-hydrogenated 0 2-equimolar 0.5

NMA (molar ratio) 0 1 0.5

D2 O (molar ratio) 15 0 7.5

H2 O (molar ratio) 0 15 7.5

Table 2: Labels and composition of the investigated NMA-trehalose-water samples. The suffix ”3-” identifies the ternary solution. The NMA and trehalose concentration is 0.57 M. Sample Label

3-deuterated 3-hydrogenated 3-equimolar 3-null 3-NMA-trehalose-8D/D2 O 3-NMA-7D -trehalose/H2 0

NMA-7D (molar ratio) 1 0 0.5 0.36 0 1

NMA trehalose-8D trehalose D2 O H2 O (molar (molar (molar (molar (molar ratio) ratio) ratio) ratio) ratio) 0 1 0 100 0 1 0 1 0 100 0.5 0.5 0.5 50 50 0.64 0.36 0.64 36 64 1 1 0 100 0 0

0

1

0

100

As it will be clear in the following, each solution, differing by its isotopic composition, represents a constraint for the computer simulation procedure used to model the experimental data. For this reason and given the relatively higher complexity of the ternary solution, more 5



samples differing by their isotopic composition were investigated in the latter case (see Table 2). Samples investigated in the experiment on the ternary solution have been prepared by using NMA, NMA-7D, trehalose (C12 H22 O11 ) and trehalose-8D (C12 D8 H14 O11 ). The latter isotopic component has been obtained by repeatedly freeze-drying a solution of trehalose in D2 O, which leads to complete substitution of the exchangeable H atoms. These samples have been dissolved in H2 O and D2 O in the proportions shown in Table 2. In particular the sample labelled as 3-null represents a neutron null-scatterer solution.

Data handling The neutron diffraction measurements, after correction for systematic errors and normalization via the GUDRUN routine 27 , yield the total interference differential scattering crosssection, F (Q). This is the linear combination of all partial structure factors, Sαβ (Q), where α, β label all atomic pairs present in the sample, each weighted by their concentration, c, and scattering length 28 , b, so that F (Q) = Σαβ cα cβ bα bβ (Sαβ (Q) − 1), where Q is the exchanged momentum in the scattering event. The Sαβ (Q) are the Fourier transforms of the pair distribution functions gαβ (r), which contain the structural information, being the density of probability that, given an atom of the pair (α) at the origin of the reference frame, the other atom (β) sits at distance r. When the number of measured differential cross-sections is lower than the number of partial structure factors of interest, the pair distribution functions can only be extracted with the aid of a computer simulation, giving the best fit of the experimental data. In the present case, the data are interpreted with the computational modeling technique called Empirical Potential Structural Refinement (EPSR) 29,30 . This is a Monte Carlo routine, which refines an interaction potential and a real space structural model of the system against the experimental data, starting from a seed potential model and a random distribution of molecules in the simulation box. Thus the more isotopic substituted samples are available, the better structural information is obtained, under the hypothesis that the isotopic sub6


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stitution does not significantly alter the structure of the solution 26 . In the present case we have measured the total neutron scattering from 3 isotopic mixtures of NMA/water and 6 of NMA/trehalose/water. The simulation boxes used in the EPSR Monte Carlo simulation in order to refine the experimental results in real space have dimensions of 31.52 ˚ A and 33.63 ˚ A for the binary and ternary solution, respectively. The first contains 50 NMA and 750 water molecules, the second 10 NMA, 10 trehalose and 1000 water molecules, giving the same density, composition and concentration as the real samples. The atomic structure of NMA and trehalose molecules is shown in Fig. 1, along with the labels hereafter assigned to the individual atomic sites. The parameters of the seed potential used to start the EPSR simulations are reported in Table 3. Table 3: Reference potential parameters used in the EPSR simulation procedure. Atoms are labelled according to Fig.1. For water atoms the standard Simple Point Charge/Extended, SPC/E, 31 potential has been used. molecule NMA

Trehalose

Water

atom label N C1 Cc Oc Cn Hn Mc O O1 O2 O3 C M H OW HW

˚) (kJ/mol) σ (A 0.71128 3.25 0.43932 3.75 0.66944 3.91 0.87864 2.96 0.71128 3.8 0 0 0 0 0.71128 3.1 0.58576 2.9 0.58576 3.1 0.71128 3.1 0.27614 3.5 0.12552 0.3 0.05 1.7 0.65 3.166 0 0

q (e) -0.55 0.58 0 -0.53 0.2 0.3 0 -0.5 -0.5 -0.5 -0.5 0.258 0 0.3005 -0.8476 0.4238

After equilibration of the simulation box (typically 1000 iterations), and potential refinement, 4500 configurations have been recorded. The final fit of the F (Q) data are shown 7



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in Fig. 2 and 3. The recorded configurations are used to calculate the radial distribution functions and the spatial density functions, SDF, discussed in the following. The SDF offer a three dimensional, visualization, around a chosen component, of the regions of space where the probability to find a particular molecule exceeds a treshold value 32 .

Results and discussion Fig. 4 shows the pair distribution functions of the NMA sites (Hn, Oc) candidate for hydrogen bonding with water sites (OW and HW) or trehalose ones (O, O3, H). It is immediately clear from this figure that the hydration of NMA sites (black and red curves in Fig. 4) does not sensibly change when trehalose is added to the solution. In particular the first peak of the gOcHW (r) is centered at ∼ 1.81 ˚ A in both solutions, and that of the gHnOW (r) moves from ∼ 2.04 ˚ A to ∼ 2.07 ˚ A on addition of trehalose. These distances, in overall agreement with literature 33 , are compatible with hydrogen bonding, although of different bond strength, being the Hn-OW bond longer and thus weaker than the Oc-HW bond. We notice a broadening of the these peaks, upon addition of trehalose: this is the signature of increased disorder, not of changed number of hydrogen bonds, Nαβ (R1 , R2 ). This latter quantity can be calculated as: Z

R2

Nαβ (R1 , R2 ) = 4πρcβ

r2 gαβ (r)dr

(1)

R1

where R1 is the minimum distance between the atom pair and R2 is the first minimum of the gαβ (r); α is the Oc or the Hn site on the NMA and β is either HW or OW. This integral gives a number of water oxygens bonded to the Hn site of the order of 0.7 in both binary and ternary solutions. The number of water hydrogen atoms bonded to the Oc site, calculated in the same way, changes from ∼ 1.5 for the binary solution to ∼ 1.6 for the ternary one. It is also evident that the first trehalose sites neighbours of the Hn and Oc atoms of NMA are found at larger distances compared to NMA-water distances. In particular the first peaks of 8


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the gHnO3 (r), and gHnO (r) are centered at r ∼ 5.0 ˚ A and r ∼ 4.2 ˚ A, respectively, that is well beyond the typical distances of atoms engaged in hydrogen bonding. Although the gOcH (r) shows a peak, compatible with hydrogen bonding at r ∼ 2.6 ˚ A, nevertheless this distance denotes a very weak interaction and the number of trehalose H sites first neighbour of an Oc site is very low (∼ 0.1). However at both Oc and Hn NMA sites, the first neighbour is a hydrogen bonded water molecule, independently on the presence of trehalose. Secondarily we notice that in the binary solution there is evidence for possible aggregation of NMA molecules (Fig. 5) not mediated by water molecules, since the gHnOc (r) function has a first neighbour peak at ∼ 2 ˚ A. Apparently trehalose inhibits such aggregation in the ternary solution, where Hn and Oc sites are pushed apart by at least 6 ˚ A. This evidence deserves further analysis as a function of trehalose and NMA concentration, as it could imply a role of trehalose in preventing protein precipitation.

Conclusions The conclusions of our work are well represented by the three-dimensional visualization of the spatial arrangement of molecules in solution shown by the SDF, plotted in Fig. 6. These show in yellow the first hydration shells of the NMA molecule in the binary (panel A) and ternary (panel B) solutions; panel B also shows in magenta the region of space occupied by trehalose in the ternary solution. Here we notice that the hydration shell of the peptide is only marginally influenced by the presence of trehalose in the ternary mixture and, more importantly, that trehalose does not substitute water molecules in the peptide hydration shell. It is indeed arranged outside the hydration shell, trapping water molecules. The ability of trehalose in trapping water molecules may be a consequence of its weak interaction with water, compared for instance with other sugars 24,34–36 . This trapping of water molecules around the peptide can prevent structural damage upon dehydration and therefore represents the key mechanism behind anhydrobiosis. Further studies at lower water content will clarify

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the role of trehalose, and in particular shed light on its ability to form a highly viscous and glassy cytoplasm, in this way protecting cells and intracellular components against droughtstress 4–6 .

Acknowledgement This work has been performed within the Agreement No.0018318 (02/06/2014) between STFC and CNR, concerning collaboration in scientific research at the spallation neutron source ISIS and with partial financial support of CNR. Beamtime awarded by ISIS under RB numbers 1510043 and 1510044 is gratefully acknowledged. The Grant of Excellence Departments, MIUR (Articolo 1, commi 314 337 Legge 232/2016) is greatly acknowledged. The authors thank S. Imberti for assistance during the experiment at ISIS.

References (1) Keilin, D. The Leeuwenhoek Lecture - The Problem of Anabiosis or Latent Life: History and Current Concept. Proc. R. Soc. London, Ser. B 1959, 150, 149–191. (2) Leopold, A. C. Membranes, Metabolism, and Dry Organisms; Cornell University Press, Ithaca, NY, 1986; pp 17–21. (3) Clegg, J. S. Cryptobiosis - a Peculiar State of Biological Organization. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 2001, 128, 613 – 624. (4) Bruni, F.; Leopold, A. C. Glass Transitions in Soybean Seed : Relevance to Anhydrous Biology. Plant Physiol. 1991, 96, 660–3. (5) Bruni, F.; Leopold, A. Pools of Water in Anhydrobiotic Organisms: A Thermally Stimulated Depolarization Current Study. Biophys. J. 1992, 63, 663–672.

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(6) Bruni, F.; Leopold, A. C. Cytoplasmic Glass Formation in Maize Embryos. Seed Sci. Res. 1992, 2, 251–253. (7) Wolfe, J. Lateral Stresses in Membranes at Low Water Potential. Funct. Plant Biol. 1987, 14, 311–318. (8) Crowe, J. H.; Crowe, L. M.; Carpenter, J. F.; Aurell Wistrom, C. Stabilization of Dry Phospholipid Bilayers and Proteins by Sugars. Biochem. J. 1987, 242, 1–10. (9) Crowe, J. H.; Crowe, L. M.; Chapman, D. Preservation of Membranes in Anhydrobiotic Organisms: The Role of Trehalose. Science 1984, 223, 701–703. (10) Carpenter, J. F.; Crowe, J. H. An Infrared Spectroscopic Study of the Interactions of Carbohydrates with Dried Proteins. Biochemistry 1989, 28, 3916–3922. (11) Crowe, L. M. Lessons from Nature: The Role of Sugars in Anhydrobiosis. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2002, 131, 505 – 513. (12) Crowe, J. H.; Crowe, L. M.; Jackson, S. A. Preservation of Structural and Functional Activity in Lyophilized Sarcoplasmic Reticulum. Arch. Biochem. Biophys. 1983, 220, 477 – 484. (13) Zentella, R.; Mascorro-Gallardo, J. O.; Van Dijck, P.; Folch-Mallol, J.; Bonini, B.; Van Vaeck, C.; Gaxiola, R.; Covarrubias, A. A.; Nieto-Sotelo, J.; Thevelein, J. M. et al. A Selaginella Lepidophylla Trehalose-6-Phosphate Synthase Complements Growth and Stress-Tolerance Defects in a Yeast tps1 Mutant. Plant Physiol. 1999, 119, 1473–1482. (14) Crowe, J. H.; Crowe, L. M.; Oliver, A. E.; Tsvetkova, N.; Wolkers, W.; Tablin, F. The Trehalose Myth Revisited: Introduction to a Symposium on Stabilization of Cells in the Dry State. Cryobiology 2001, 43, 89 – 105. (15) Crowe, J. H.; Carpenter, J. F.; Crowe, L. M. The Role of Vitrification in Anhydrobiosis. Annu. Rev. Physiol. 1998, 60, 73–103. 11



(16) Green, J. L.; Angell, C. A. Phase Relations and Vitrification in Saccharide-Water Solutions and the Trehalose Anomaly. J. Phys. Chem. 1989, 93, 2880–2882. (17) Sampedro, J. D.; Uribe, S. Trehalose-Enzyme Interactions Result in Structure Stabilization and Activity Inhibition. The Role of Viscosity. Mol. Cell. Biochem. 2004, 256, 319 – 327. (18) Sola-Penna, M.; Meyer-Fernandes, J. R. Stabilization against Thermal Inactivation Promoted by Sugars on Enzyme Structure and Function : Why Is Trehalose More Effective Than Other Sugars? Arch. Biochem. Biophys. 1998, 360, 10–14. (19) Cottone, G.; Ciccotti, G.; Cordone, L. Proteine-Trehalose-Water Structures in Trehalose Coated Carboxy-Myoglobin. J. Chem. Phys. 2002, 117, 9862–9866. (20) Belton, P.; Gil, A. M. IR and Raman Spectroscopic Studies of the Interaction of Trehalose with Hen Egg white Lysozyme. Biopolymers 1994, 34, 957–61. (21) Corradini, D.; Strekalova, E. G.; Stanley, H. E.; Gallo, P. Microscopic Mechanism of Protein Cryopreservation in an Aqueous Solution with Trehalose. Sci. Rep. 2013, 3, 1218. (22) Guo, N.; Puhlev, I.; Brown, D. R.; Mansbridge, J.; Levine, F. Trehalose Expression Confers Desiccation Tolerance on Human Cells. Nat. Biotechnol. 2000, 18, 168. (23) Cunha, A. V.; Salamatova, E.; Bloem, R.; Roeters, S.; J. Woutersen, S.; Pshenichnikov, M. S.; Jansen, T. L. C. Interplay between Hydrogen Bonding and Vibrational Coupling in Liquid N-Methylacetamide. J. Phys. Chem. Lett. 2017, 8, 2438–2444, PMID: 28510458. (24) Bruni, F.; Di Mino, C.; Imberti, S.; McLain, S. E.; Rhys, N. H.; Ricci, M. A. Hydrogen Bond Length as a Key To Understanding Sweetness. J. Phys. Chem. Lett. 2018, 9, 3667–3672, PMID: 29920095. 12


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(25) for information: https://www.isis.stfc.ac.uk/Pages/sandals.aspx. (26) McLain, S. E.; Imberti, S.; Soper, A. K.; Botti, A.; Bruni, F.; Ricci, M. A. Structure of 2 molar NaOH in Aqueous Solution from Neutron Diffraction and Empirical Potential Structure Refinement. Phys. Rev. B 2006, 74, 094201. (27) Soper, A. K. In RAL Rep. RAL-TR-2011-013 ; Rutherford Appleton Laboratory, S. T. F. C., Ed.; 2011. (28) Sears, V. F. Neutron Scattering Lengths and Cross Sections. Neutron News 1992, 3, 26–37. (29) Soper, A. Empirical Potential Monte Carlo Simulation of Fluid Structure. Chem. Phys. 1996, 202, 295–306. (30) Soper, A. K. Partial Structure Factors from Disordered Materials Diffraction Data: An Approach Using Empirical Potential Structure Refinement. Phys. Rev. B 2005, 72, 104–204. (31) Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. The Missing Term in Effective Pair Potentials. J. Phys. Chem. 1987, 91, 6269–6271. (32) Svishchev, I. M.; Kusalik, P. G. Structure in Liquid Water: A Study of Spatial Distribution Functions. J. Chem. Phys. 1993, 99, 3049–3058. (33) Gao, J.; Freindorf, M. Hybrid ab Initio QM/MM Simulation of N-Methylacetamide in Aqueous Solution. J. Phys. Chem. A 1997, 101, 3182–3188. (34) Pagnotta, S. E.; McLain, S. E.; Soper, A. K.; Bruni, F.; Ricci, M. A. Water and Trehalose: How Much Do They Interact with Each Other? J. Phys. Chem. B 2010, 114, 4904–4908, PMID: 20297794.

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(35) Maugeri, L.; Busch, S.; McLain, S. E.; Pardo, L. C.; Bruni, F.; Ricci, M. A. StructureActivity Relationships in Carbohydrates Revealed by their Hydration. Biochim. Biophys. Acta, Gen. Subj. 2017, 1861, 1486 – 1493, Recent Advances in Bionanomaterials. (36) Soper, A. K.; Ricci, M. A.; Bruni, F.; Rhys, N. H.; McLain, S. E. Trehalose in Water Revisited. J. Phys. Chem. B 2018, 122, 7365–7374.

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Graphical TOC Entry

A

B

water

NMA

trehalose

graphic.pdf

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A

B Oc N C1 Mc

Cc Cn Hn

Figure 1: (Color online) Atom labeling for the NMA (A) and trehalose (B) molecules used in EPSR analysis and in the pair distribution functions reported hereafter. The carbon atoms on the NMA molecule are labeled Cn, C1 and Cc in order to evidence their different environments; N and Hn label the NH site components, the oxygen site is labeled Oc in order to distinguish it from the oxygen sites of trehalose; all hydrogens bonded to carbon sites are labeled Mc. In the trehalose molecule, all hydrogens bonded to oxygens are labeled H, while the label M is assigned to those bonded to carbons (all labeled C). The bridging oxygen atom between the two glucose rings of trehalose is labeled O1, the oxygen atoms within each glucose ring are labeled O2, the oxygen atoms attached to CH2 groups are labeled O3 and the oxygen atoms bonded directly to the ring carbon atoms are labeled O. Water sites (not shown in this figure) are labeled OW and HW.

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1.2 1.0 0.8 0.6 F(Q)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.4

,

2-deuterated

,

2-hydrogenated

,

2-equimolar

0.2 0.0 -0.2 -0.4 0

5

10

15

20

25

30

-1

Q (Å ) Figure 2: Differential scattering cross-section, F(Q), of the binary solution: data (solid lines) and EPSR fit (dashed lines).

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2.5

, , , , , ,

2.0

F(Q)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1.5

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3-deuterated 3-hydrogenated 3-null 3-NMA-trehalose-8D in D2O 3-equimolar 3-NMA-7D-trehalose in H20

1.0 0.5 0.0 0

5

10

15

20

25

30

-1

Q (Å ) Figure 3: Differential scattering cross-sections, F(Q), of the ternary solution: data (solid lines) and EPSR fit (dashed lines).

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0

1

2

3

4

5

6

7

6

7

3.5 NMA-water binary mixture NMA-water ternary mixture

3.0

NMA Oc -Trehalose H ternary mixture ,

NMA Hn -Trehalose oxygens ternary mixture

2.5 g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hn-O3

2.0

Hn-O Hn-OW

1.5 Oc-H

1.0 0.5 Oc-HW

0.0 0

1

2

3

4

5

r (Å) Figure 4: (Color on line) The pair distribution functions of the hydrophilic sites of NMA, namely Oc and Hn, and water or trehalose atoms. Data for the binary NMA-water solution are reported in black; the same distributions for the ternary solution are reported in red. As far as the NMA-trehalose distribution functions are concerned, the gOcH (r) is reported in green, the gHnO (r) and gHnO3 (r) are reported in blue and magenta, respectively. The dashed vertical lines evidence the position of the maximum of the H-bond peaks (same color code as for the radial distribution functions).

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1.0

Hn-Oc binary mixture Hn-Oc ternary mixture

0.8

g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.6 0.4 0.2 0.0 0

2

4

6

8

10

12

r (Å) Figure 5: (Color on line) The radial distribution functions of the Hn and Oc sites of NMA, showing aggregation of molecules in the binary solution (black curve), with a peak at ∼ 2 ˚ A. In the ternary solution (red curve) the minimum distance between Hn and Oc sites on distinct NMA molecule is beyond 6 ˚ A.

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A

B

Figure 6: (Color on line) The spatial distribution functions around a NMA molecule: A: The yellow region represents the area occupied by water molecules in the first hydration shell of NMA in the binary solution. B: The first hydration shell of NMA in the ternary solution is represented in yellow, while the region occupied by the trehalose first neighnours is highlighted in magenta. For the water distribution the shown region encloses 15% of the first neighbour water molecules in the distance range 0−6.6 ˚ A from the central NMA molecule. The magenta region encloses 15% of the first neighbours trehalose molecules in the distance range 0−10 ˚ A. 21


Protection against Dehydration: a Neutron Diffraction Study on

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