More Is Different: Experimental Results on the Effect of Biomolecules

Letter pubs.acs.org/JPCL

More Is Different: Experimental Results on the Effect of Biomolecules on the Dynamics of Hydration Water Lucia Comez,†,‡ Laura Lupi,† Assunta Morresi,§ Marco Paolantoni,§ Paola Sassi,§ and Daniele Fioretto*,†,⊥ †

Dipartimento di Fisica and ‡IOM-CNR c/o Dipartimento di Fisica, Università di Perugia, Via Pascoli, I-06123 Perugia, Italy Dipartimento di Chimica and ⊥Centro di Eccellenza sui Materiali Innovativi Nanostrutturati (CEMIN), Università di Perugia, Via Elce di Sotto 8, I-06123 Perugia, Italy

§

ABSTRACT: Biological interfaces characterized by a complex mixture of hydrophobic, hydrophilic, or charged moieties interfere with the cooperative rearrangement of the hydrogen-bond network of water. In the present study, this solute-induced dynamical perturbation is investigated by extended frequency range depolarized light scattering experiments on an aqueous solution of a variety of systems of different nature and complexity such as small hydrophobic and hydrophilic molecules, amino acids, dipeptides, and proteins. Our results suggest that a reductionist approach is not adequate to describe the rearrangement of hydration water because a significant increase of the dynamical retardation and extension of the perturbation occurs when increasing the chemical complexity of the solute.

SECTION: Biophysical Chemistry and Biomolecules

W

of choice to answer this question because relaxation of anisotropic polarizability occurs in the 100 GHz frequency region, that is, faster than the exchange rate between bulk and hydration molecules. Moreover, EDLS extends over the whole frequency region covered by solute and solvent diffusional dynamics, so that it gives both relaxation times and relative fractions of hydration and bulk water.17 The systems under investigation include two representative small hydrophobic molecules (TBA and TMAO), one amino acid (lysine), and three amphiphilic model peptides, namely, a model of a hydrophilic backbone, N-acetyl-glycine-methylamide (NAGMA), a more hydrophobic peptide, N-acetyl-leucinemethylamide (NALMA, ref 17), and the homologue N-acetylleucine-amide (NALA), which differs from NALMA by a methyl group at the terminal amide group. Given their lengths and curvatures, these peptides are thought10 to be more suitable as “protein models” than single amino acids, without backbone chemical heterogeneity and interactions. Moreover, in order to obtain a more general picture, the present data are compared with those previously obtained on aqueous solutions of sugars18,19 that are representative of simple hydrophilic systems and of lysozyme, the prototype of small globular proteins.20 Because the values of hydration number are known to be concentration-dependent,19 we performed measurements at

ater greatly affects structure, dynamics, and functionality of biomolecules participating in different processes in a wide range of length and time scales. On the other hand, water experiences a quite complex environment, which influences the cooperative restructuring process of the H-bond (HB) network. Trying to understand the mutual influence of water and biomolecules, a noticeable experimental and numerical effort has been recently devoted to the study of small molecules, which mimic the effect of portions of biomacromolecules, on surrounding water. Hydrophilic1 and amphiphilic solutes,2−4 with more or less pronounced hydrophobic parts,5 amino acids,6,7 and peptides8−10 up to proteins11 have been studied by different techniques, like NMR,8 dielectric spectroscopy,3,12,13 THz spectroscopy,7 neutron scattering,10,11 OKE,2 2D IR spectroscopy,9 polarized,14 and depolarized light scattering,15 and by numeric4 and analytic models.6,16 The great majority of these investigations gives evidence of a retardation of hydration water with respect to the bulk, the extent and the spatial extension of the effect being still very debated. An important step to give a rationale of the mutual influence among water and complex biological matter is to understand whether and to what extent the results obtained in water solutions of small molecules can be extrapolated to the case of biomacromolecules, like proteins, and even cells and tissues. To this respect, a relevant question arises, Is it possible to understand the ef fect of a macromolecule on surrounding water by a reductionist approach, that is, as the simple addition of the effects induced by its small parts? Among spectroscopic techniques, extended frequency range depolarized light scattering (EDLS)17 can be seen as a method © XXXX American Chemical Society

Received: February 17, 2013 Accepted: March 20, 2013

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frequency one, located at several tens of GHz (∼30 GHz for the amino acid and the peptide, ∼40 GHz for fructose, and ∼50 GHz for TMAO), and a high-frequency one, at around 2−4 THz, where the boson peak of condensed systems is usually located,21 here attributed to collective internal vibrational modes of the solute.20 The low-frequency region may comprise contributions from hydration water and cross solute−water terms. In the following, we attribute this contribution to the relaxation of hydration water alone, guided from results of the detailed MD simulation performed on water−carbohydrates solutions.18 Figure 2 shows that both retardation and a spectral amplitude increase passing from small hydrophilic and hydrophobic molecules to amino acids and peptides. Retardation ξ, that is, the ratio between hydration and bulk water relaxation times, can be estimated by measuring the distance between the relaxation peaks in Figure 2 and the dashed line, located at ∼230 GHz, representing the maximum of the relaxation function of bulk water. In this way, retardation values of about 5 for TBA, TMAO, and sugars and about 8 for amino acids and peptides are found. Connected with this increasing retardation, the larger area of EDLS peaks gives evidence for a much larger amount of water slowed down by amino acid and peptide molecules with respect to sugar and hydrophobic solutes. These results, independent from any fitting procedure, are a clear indication of the role of the different solutes in determining the strength and extension of dynamic perturbation on hydration water. To deepen our discussion, a quantitative analysis of the spectra is required, giving both the extent and spatial extension of the disturbance induced by the solute. In line with the data analysis procedure developed in previous works,17,19,20,22 EDLS spectra are interpreted as the sum of two DHO components for the intermolecular vibration of water molecules (vibrations region in Figure 1) and two Cole Davidson (CD) relaxation functions for the contribution of bulk-like and hydration water. The relaxation times and amplitudes of both bulk and hydration water are left free during the fitting procedure and are used to estimate the values of ξ and Nh reported in Table 1.

different concentrations for each sample. For a more meaningful comparison among different systems, here, we report measurements taken at relatively low and similar concentrations, namely, a ∼90 mg/mL TBA−water solution, a ∼130 mg/mL TMAO−water solution, a ∼130 mg/mL lysine−water solution, and ∼100 mg/mL NALMA−, NALA−, and NAGMA−water solutions. Depolarized light scattering from these systems has been performed at ambient temperature over a broad frequency domain by the combined use of interferometric and dispersive devices.15 The resulting susceptibility spectra, relative to some of the investigated samples, are reported in Figure 1.

Figure 1. Imaginary part of the susceptibility of pure water and water solutions at 20 °C, measured by EDLS.

To better emphasize the modifications induced by the solutes to the spectrum, in Figure 2, we report the solventrotation-free (SRF) spectra of four representative systems, after subtraction of the pure water signal and of the solute rotational contribution, estimated by the procedure already described in ref 17. Two distinct features can be appreciated, a low-

Table 1. Molecular Weight, Volume, Hydration/Bulk Retardation Factor ξ, Hydration Number Nh, and Effective Thickness of the Perturbed Hydrations Shell h for Each Solutea m.w. (Da) TBA TMAO glucose

74.12 75.11 180.16

fructose trehalose

180.16 342.3

sucrose lysine NAGMA NALA NALMA lysozyme

Figure 2. SRF spectra of the indicated water solutions. The solvent spectra and the rotational relaxation contribution of the solute have been subtracted from the total spectra to emphasize hydration water and vibrational internal modes of the solute molecules.

342.3 146.2 130.1 172.2 186.3 14307

V (Å3 )

ξ

86.6 83.6 150.4 (166) 150.3 281.6 (330) 281.5 146.0 125.0 165.6 191.8 (33510)

4−5 4−5 5−6

14 12 16

5−6 5−6

15 25

5−6 7−8 7−8 7−8 7−8 7−8

27 32 48 57 61 2300

Nh

h (Å) 2.2 2.1 2.0 1.9 1.9 2.2 2.1 2.3 3.1 4.1 4.2 4.3 9.0

a

van der Waals volumes are generally reported, apart from those between parentheses that are obtained by light scattering measurements.20,23,24.

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mated by the convolution product of dynamic structure factors S(q0,ω) ⊗ S(q0,ω), where q0 is the wavenumber corresponding to the maximum of the static structure factor. Conversely, BLS reveals the dynamic structure factor S(q0,ω) at much smaller wavenumbers,15 comparable with that of visible light. The convolution procedure and the q dependence of the relaxation times, which is typical of all liquids close to q0,28 may be the reason for the discrepancy between relaxation times and retardations obtained by the two techniques. The number of retarded molecules obtained from the relaxation strength of the hydration water band in TBA and TMAO is 12−14, corresponding to an average thickness of about 2.1−2.2 Å. This means that the perturbation induced by these small molecules is localized within the first geometric hydration shell of thickness ρ−1/3 ≈ 3.1 Å around the solute. This result is also consistent with that found by dielectric spectroscopy3 and by MD simulations.29 Sugar molecules are also found to produce a little disturbance in the dynamic properties of water. In fact, though mono- and disaccharides occupy volumes 2−4 times larger than those of TBA and TMAO and one might expect a much larger effect induced on the network of HBs, our experiments give evidence of a retardation only slightly larger than that induced by small hydrophobic molecules, and h ≈ 2 Å, comparable to that of smaller molecules. Recently, the concentration of hydroxyl groups has been found to be the parameter guiding sugar− water interactions, without appreciable difference between mono- and disaccharides, all inducing about 3.3 water molecules per sugar hydroxyl group to be part of the hydration shell.19 The small number of perturbed water molecules can be thus attributed to the presence of hydroxyl groups uniformly distributed on sugar’s surface that fit very well into the tetrahedral structure of the water network, only slightly perturbing its structure and dynamics. An important discontinuity is found passing from TBA, TMAO, and sugars to diluted aqueous solutions of amino acids and dipeptides, showing a retardation factor of about 8 affecting 120−200 surrounding water molecules, corresponding to a spatial extension that is twice that perturbed by sugars, that is, h ≈ 4 Å. It is also evident that this strong effect is not a matter of dimension of the solute because amino acids and peptides are smaller than disaccharides. Trying to rationalize this result, it must be mentioned that the hydrophilic (HB acceptors) carbonyl groups of dipeptides and amino acids are known to have a pronounced effect on the HB dynamics of water.6,9 Moreover, it has been argued for a long time that whether “bound” water is present in polypeptides, it has to be close to the peptide bond.30 Notwithstanding this, the extent of the perturbation found by EDLS is quite peculiar and can be hardly attributed to the presence of a single chemical group in the molecule. In fact, a HB acceptor is also present in TMAO, which does not show the strong effect in hydration water reported for amino acids and peptides. Recent MD simulations of NALMA31 reported dielectric relaxation anomalies, which were attributed to frustration in the water network arising from the amphiphilic chemistry of the peptide that does not allow it to reorient on the picosecond time scale of bulk water motions. To this respect, our results suggest that the amphiphilic nature of the solute alone is not so crucial because also NAGMA, a model of the hydrophilic backbone, produces a similar effect. The peculiar influence of peptides on surrounding water molecules possibly resides in a nontrivial mixture of effects related to the presence of polar groups, amphiphilic character,

To give a semiquantitative picture, which helps to compare the effects induced on water by the different solute molecules, the average spatial extension h of perturbed water molecules surrounding the solute has been estimated from the value of Nh by means of a simple geometric model. In the case of almost spherical molecules, the effective value of h is calculated from the relationship Nh = 4π(h3/3 + h2r + hr2)ρ, where ρ is the number density of neat water and r is the average radius of the solute. Error bars are not reported for h because it is an effective number, whose value is based on the rather crude spherical approximation. Notwithstanding this, the strong observed variation of h is out of any possible approximation and experimental error. The values of the retardation ratios are also reported in Figure 3. To complete the picture, it is interesting to notice that

Figure 3. Slowing down factors of aqueous solutions including small hydrophobes, mono- and disaccharides, amino acids, dipeptides, and the globular protein lysozyme as solutes.

measurements performed in some of these samples as a function of solute concentration (not reported here) have shown a rather constant value of ξ, so that the results in Figure 3 can be safely considered as concentration-independent, within experimental errors. It can be seen that retardation values are in agreement with what was visually discerned in the SRF spectra of Figure 2, corroborating the truthfulness of the fitting procedure. Small, prevalently hydrophobic molecules (TBA and TMAO) show the smallest retardation effect. Recently, a model has been proposed that suggests the retardation of water around hydrophobic molecules to arise from the lack of volume available for the diffusion, which reduces the activation entropy in rotational jumps.25 Brillouin light scattering (BLS) measurements have confirmed the predictions of the model, showing that it applies not only to rotational reorientations but also to density fluctuations.14 Here, EDLS results essentially confirm this picture because the retardation induced by hydrophobic molecules is the smallest of the series, even if a slightly higher value for ξ is found with respect to BLS, which might be attributed to the physically distinct processes revealed by the two techniques. In fact, we have recently shown that the water contribution to the anisotropic polarizability of water−sugar solutions is dominated by interaction-induced contributions.18 Indeed, in previous studies, the low-frequency Raman spectra of both simple liquids26 and solutions27 have been interpreted in terms of dipole-induced-dipole (DID) interaction-induced scattering, and the spectrum has been successfully approxi1190

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Notes

frustration, and so forth, rather than in the simple addition of the effects related to hydrophilic and hydrophobic parts of the molecules. The greater chemical complexity seems to be the correct key to interpreting the greater effect of amino acids and peptides on the hydration water. Important evidence of the role of complexity is found in the further discontinuity in hydration properties that is found when passing from peptides to lysozyme. In fact, though the average relaxation time of hydration water around the protein is close to that of water around amino acids and dipeptides, the perturbation here extends upon a considerably wider region, reaching h ≈ 9 Å, almost three times the thickness of one geometric shell. This result seems to contradict the idea that the perturbation induced by a biological interface is necessarily of short-range, essentially limited to the first coordination shell. This idea, suggested by the attitude of liquid water to minimize the effect of solutes on its HB network structure due to its very high cohesive energy density,32 was confirmed by single water molecule rotational dynamics probed by NMR and MD simulations.32,33 The apparent contradiction of our results with this idea can be tentatively explained by considering that NMR probes the single water molecule rotational motion while EDLS measures the power spectrum of a four-body correlation function and is sensitive to the interaction effects induced among different molecules. For this reason, it seems reasonable that EDLS is more sensitive to long-range effects, affecting the collective dynamics of water molecules, even in conditions where static properties or local rotations are little perturbed. Collective effects induced by large protein surfaces on surrounding water have been, indeed, suggested by THz spectroscopy.34 More recently, analytic models and MD simulations35,36 inferred that large hydrophobic regions significantly affect the interfacial water structure and that unsatisfied HBs at the water−solute interface can cause an energetic push for in-plane orientations of the water dipoles, promoting the development of ferroelectric hydration shells around proteins, propagating up to 3−5 water diameters into the bulk. Even though these models predict a larger effect in the case of redox-active proteins than that in lysozyme, they provide a rather new and promising point of view for their ability to capture peculiar dynamic properties emerging in the biological mesoscale not included either in the molecular or in the mean field approaches. In summary, the results of EDLS experiments on water solutions of small hydrophobics, sugars, amino acids, peptides, and proteins let us believe that, in general, it is not possible to explain the influence of a biomacromolecule on surrounding water in terms of its small parts. In fact, we have seen that by increasing the complexity of the solute, new properties of the hydration shell emerge, which are not explained by a mere superposition of the effects induced by the single parts. Both the retardation factor and the hydration number, that is, the average number of molecules whose dynamics are affected by the solute, are found to strongly depend on the solute, giving support to the idea that more is dif ferent, even for relatively small systems, like biomolecules surrounded by water. These experimental results challenge future theoretical developments to a deeper understanding of the mutual dependence of the properties of water and its biomolecular partners.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.P. acknowledges support from the Italian Ministry of Education, University and Research (MIUR) under the PRIN 2010-2011 project.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: daniele.fioretto@fisica.unipg.it. 1191

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