Letter pubs.acs.org/JPCL
Modulation of the Hydrogen Bonding Structure of Water by Renal Osmolytes Pramod Kumar Verma,†,‡ Hochan Lee,†,‡ Joon-Young Park,†,‡ Joon-Hyung Lim,†,‡ Michał Maj,†,‡ Jun-Ho Choi,† Kyung-Won Kwak,§ and Minhaeng Cho*,†,‡ †
Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Korea University, Seoul 136-701, Republic of Korea ‡ Department of Chemistry, Korea University, Seoul 136-701, Republic of Korea § Department of Chemistry, Chung-Ang University, Seoul 156-756, Republic of Korea S Supporting Information *
ABSTRACT: Osmolytes are an integral part of living organism, e.g., the kidney uses sorbitol, trimethylglycine, taurine and myo-inositol to counter the deleterious effects of urea and salt. Therefore, knowing that the osmolytes’ act either directly to the protein or mediated through water is of great importance. Our experimental and computational results show that protecting osmolytes, e.g., trimethylglycine and sorbitol, significantly modulate the water H-bonding network structure, although the magnitude and spatial extent of osmolyte-induced perturbation greatly vary. In contrast, urea behaves neutrally toward local water H-bonding network. Protecting osmolytes studied here show strong concentration-dependent behaviors (vibrational frequencies and lifetimes of two different infrared (IR) probes), while denaturant does not. The H-bond donor and/or acceptor (OH/NH) in a given osmolyte molecule play a critical role in defining their action. Our findings highlight the significance of the alteration of H-bonding network of water under biologically relevant environment, often encountered in real biological systems.
A
Scheme 1. Structures of the Renal Osmolytes Studied in the Present Work
lmost all living organisms are equipped with osmolytes, naturally occurring small organic molecules, to cope with harsh environmental conditions such as potentially harmful fluctuations in temperature, pressure and solution composition.1,2 The best-known example for osmotic stress under normal conditions in humans is the kidney.3,4 The mammalian kidney accumulates taurine, myo-inositol, sorbitol, trimethylglycine (TMG) and glycerophosphocholine and uses them to counteract the deleterious effects of high renal urea (up to 5.4 M in water-independent rodent) and salt concentrations.3,5 These osmolytes can be grouped into two classes based on their chemical structures; those having multiple H-bonding sites (OH or NH), e.g., urea, sorbitol, myo-inositol (Scheme 1), and those without any H-bond donating site such as TMG (neither OH nor NH). It is known that these osmolytes are typically accumulated in the intracellular environment at relatively high concentrations (highest concentrations in tissue culture are 1.1 molkg−1 for sorbitol and 0.2 molkg−1 for TMG) to maintain the intracellular proteins in a soluble form, but how they act still remains a hotly debated question.5,6 It is extremely important to understand the underlying mechanisms of how they stabilize (or destabilize) proteins/ enzymes in a living organism. Do they induce a structural modification of water or act directly on proteins? If they do modulate the properties of water molecules, does the presence of multiple H-bond donor and/or acceptor sites (OH or HN) © 2015 American Chemical Society
in osmolytes play a critical role? To provide conclusive answers, we use femtosecond (fs) IR pump−probe (PP) measurement Received: May 25, 2015 Accepted: June 29, 2015 Published: June 29, 2015 2773
DOI: 10.1021/acs.jpclett.5b01087 J. Phys. Chem. Lett. 2015, 6, 2773−2779
Letter
The Journal of Physical Chemistry Letters
Figure 1. OD stretch of HDO and azide stretch of HN3 in aqueous sorbitol (A and C) and TMG (B and D) solutions. The details of background subtraction are shown in Figure S1 of the Supporting Information. ΔPeak position and Δfwhm (full width at half-maximum) of Pseudo-Voigt fits (see Figure S2 of the Supporting Information) to the FTIR spectra of the OD (E and F) and azide (G and H) bands as a function of osmolyte concentrations.
corresponds to an increase in the population of osmolyte− water H-bond or stronger water−water H-bonds, whereas the changes in the blue-side come from a reduction in the number or weakening of H-bond network in osmolyte solutions. Among all the studied osmolytes, the largest red-shift (17 cm−1) is observed in sorbitol (Figure 1E); note that Groot at al.7 did not find any clear peak shift in OD band of sorbitol solution, which is because, we believe, they used a different approach to background subtraction. Solute−Water (NNN−H2O) Interaction. Similar to OD band, azide band shows a substantial red-shift (Figure 1C,D,G) on addition of osmolytes except urea, which is induced by the change in the population of the NNN−H2O H-bond rather than its strength. Note that the azide stretching frequency maximum shows no remarkable dependency on solvent polarity but is highly correlated to the number and orientation of Hbond donors.15,16 For example, its frequency in water is higher than any other H-bond donating solvent including solvents that are stronger H-bond donors.15 Thus, the red-shift of the azide peak frequency provides direct indication that TMG, which by itself is unlikely to form H-bonds with the NNN group, can decrease the NNN−H2O H-bond population while enhancing the TMG−H2O at the expense of water−water. TMG causes a larger red-shift of the azide band than that of OD band suggesting that TMG−H2O partnership is largely increased over others, but the increase in the average H-bond strength is moderate. On the other hand, sorbitol causes a larger red-shift of OD band than that of azide band, signifying that sorbitol− H2O interaction is slightly favored, but the increase in the average H-bond strength is significant. Note that, although sorbitol causes a smaller red-shift of the azide band than that of OD band, the increase in fwhm is almost same for both the bands. This is due to the diversity of H-bond forming capacity of sorbitol. Sorbitol having six hydroxyl groups can form an Hbond with NNN apart from NNN−H2O H-bonds, whereas, in the case of TMG (zwitterion), there exist mainly NNN−H2O
along with molecular dynamics (MD) simulation. Our approach, which differentiates from the previous works, is to use two different IR probes. All the previous studies in this research field considered the OD stretch mode of the HDO dissolved in osmolyte solutions, where HDO forming a H-bond with water as well as osmolyte molecules has been used as the direct probe.7−10 However, HDO provides information on water structure only from the water’s point of view and does not provide any direct information on how a given osmolyte dictates water’s choice to be H-bonded either with another water or a third molecular component (e.g., protein or solute) in solution. Moreover, the vibrational lifetime of the OD stretch mode was found to be negligibly dependent on sorbitol7 and urea8 concentrations, but these results seem to be inconsistent with the solvation behaviors of osmolytes.11−13 That is to say, all protecting osmolytes (like sorbitol) follow a very strongly concentration-dependent solvation behavior while denaturant (urea) does not.11 In the present study, we have employed an additional (spectator) IR probe, i.e., hydrazoic acid (HN3), which provides information from the dissolved solute’s point of view and allows a direct assessment of how water’s choice of H-bonding is altered by osmolyte−water and osmolyte−osmolyte interactions. Our dual-IR-probe approach thus enables us to have stereoscopic and complementary views on osmolyte-induced changes of water structure. Linear Spectra. The normalized absorbance spectra of HDO and HN3 in liquid water and in various osmolyte solutions of different concentrations are shown in Figure 1. All the osmolytes except urea cause a red-shift of OD band with significant line-broadening observed on the red-side, while weaker narrowing is seen on the blue-side of the spectrum. Such asymmetric (nonmonotonic) frequency component shifts in the low- and high-frequency sides of the FTIR spectrum result from redistribution of equilibrium H-bond numbers and H-bonding strengths.14 The broadening on the red-side 2774
DOI: 10.1021/acs.jpclett.5b01087 J. Phys. Chem. Lett. 2015, 6, 2773−2779
Letter
The Journal of Physical Chemistry Letters
Figure 2. Site−site rdf of Ow−Hw for different concentrations of sorbitol (A), TMG (B) and urea (C). First maximum of Ow−Ow rdf (D) and Hbond number (E) as a function of various osmolyte concentrations. The details of MD simulation, a few snapshots (Figure S3), and the convergence of the Ow−Hw rdf (Figure S4) are provided in the Supporting Information.
Figure 3. Isotropic IR PP signal of water, sorbitol (4 M), TMG (4 M), and urea (8 M) in isotopically diluted water (A−D) and in hydrazoic acid solution (E−H). The data (isotopically diluted water) represent fits to a model proposed by Bakker et al. that takes into account the effects of ingrowing heating contribution (the details of the fit and uncorrected IR PP spectra are provided in Figure S6 of the Supporting Information).19,20 The isotropic IR PP signal of the azide band in all the osmolytes shows negligible in-growing heating contribution.
TMG (neither OH nor NH present) increases the first peak of the Ow−Hw rdf considerably, but the second peak increases comparatively marginally. This means that the impact of TMG on water structure is limited to the immediate first hydration layer. The magnitude of the increase in Ow−Ow rdf (Figure 2D) on addition of TMG is smaller compared to that of the same concentration of sorbitol, though both TMG and sorbitol decrease the H-bond number (Figure 2E). This decrease in Hbond number may not be due to larger excluded volume of osmolytes, because addition of NaCl also significantly decreases the H-bond number, though the corresponding ions do not possess large excluded volumes.17 Other protecting osmolytes (myo-inositol and taurine) also follow a similar trend, but their
H-bonds. To confirm these insights on the action of osmolyte, MD simulations were run for all the osmolyte solutions in a wide range of concentrations. In Figure 2A−C, the calculated radial distribution functions (rdf) between Ow (water O atom) and Hw (water H atom) are plotted. Sorbitol increases both the first and second peaks of Ow−Hw rdf by the same magnitude (Figure 2A). This indicates that sorbitol having multiple H-bonding sites (OH) influences not only the first hydration but the second hydration layer as well. This extended effect on water structure is clearly reflected in the Ow−Ow rdf (Figure 2D) of which first maximum increases almost twice compared to that of bulk water. These observations show the diffusive and long-range effect of sorbitol on H-bonding structure of water molecules. On the contrary, 2775
DOI: 10.1021/acs.jpclett.5b01087 J. Phys. Chem. Lett. 2015, 6, 2773−2779
Letter
The Journal of Physical Chemistry Letters
Figure 4. Vibrational lifetime of the OD and azide bands as a function of various osmolyte concentrations (A). Concentration dependence of τrot for the center of the OD bands (2518 cm−1) and azide band (2147 cm−1) for aqueous solutions of myo-inositol, sorbitol, taurine, trimethylglycine and urea (B). Individual fitting of the anisotropy data for various osmolytes is shown in Figures S7, S8, S9, S10 and S11 of the Supporting Information.
lifetime of OD stretch,21,22 which is almost independent of sorbitol concentration (Figure 4A). In the sorbitol−water system, there exists sorbitol−sorbitol, sorbitol−water, and water−water H-bonds. The results of FTIR measurements combined with MD simulations suggest an enhancement of sorbitol−water participation while keeping a considerable population of water−water still intact (Figure 1A, the blue arrow corresponding to weaker narrowing of the blue side of the OD spectrum). This might explain why even at a very high concentration of sorbitol, the VER of OD vibration is as rapid as that in bulk water. Similar to sorbitol, myo-inositol, taurine, TMG and urea have negligible effect on vibrational lifetime of OD band (Figure 4A). Hence, the vibrational lifetime of the direct probe (OD stretch vibration) does not seem to be a useful probe for monitoring local changes in the water structure in the presence of osmolytes. Unlike OD, vibrational lifetime (2.6 ps; average of 0−1 and 1−2 transitions)23 of the azide band significantly increases with osmolyte (except urea) concentration (inset of Figure 4A), which reflects HN3’s unique sensitivity to local H-bonding environment.15,24,25 For example; its lifetime increases 2-fold in going from water to methanol.23 In a nanometer-sized reverse micelle, its lifetime in the interfacial region, where the availability of H-bond donor to an azide group is scarce, is significantly longer than that in the core region of bulk type water environment.26 The increase in the lifetime with increasing osmolyte concentration clearly indicates that the NNN−H2O H-bond population is significantly reduced due to the change in local H-bonding environment. In a binary mixture of osmolyte and water, the water has the choice to be H-bonded either to another water or an osmolyte. As the concentration of osmolyte increases, water’s choice of being Hbonded either with another water or osmolyte becomes important. The red-shift of the azide band with the addition of osmolyte suggests that water favors osmolyte more than HN3. Hence, the decreased availability of H-bonding partner toward HN3 is manifested by its increased lifetime in the presence of osmolytes. At all concentrations, TMG is more effective in perturbing water−water H-bonding partnership compared to sorbitol, as seen by the increasing vibrational lifetime of the azide. With the addition of TMG, the tetrahedral network of water is partially broken,27 and TMG takes away water from NNN−H2O H-
much lower solubility ( sorbitol ≫ urea) and transfer free energies of the peptide backbone (the main contribution to the proteinfolding efficacy).11,37,38 Here, we have demonstrated that the dual IR-probe approach with fs IR PP and MD simulation methods are exceptionally useful to elucidate the underlying
bonding species and hence the dissipation of vibrational excitation energy becomes slower. Sorbitol being a linear and flexible molecule with six available hydroxyl groups can form Hbonds among themselves as well as to other molecules present in the solution. Such diversity in H-bonding capability seems to compensate for the H-bonding partner and hence the increase of the vibrational lifetime of azide (sorbitol) is small when compared to TMG at the same concentration. Adding osmolyte does not always mean to break/form a H-bond or to change the solute−water population as seen in the case of urea. Urea seems to be very compatible with water in a sense that water finds both urea and water to be an equally suitable partner for Hbonding, even up to very high concentration (8 M) as seen in negligible shift of the OD and azide peak frequency. The negligible dependence of the lifetime of both OD and azide for urea/water solutions corroborates well with the FTIR measurement and MD simulation as well as to previous studies.8,28,29 Orientational Relaxation. A change in water structure should be reflected in the water reorientation dynamics. TMG possesses a bulky trimethyl group like in trimethylamine-Noxide (TMAO), but the latter is smaller in length. At lower TMG concentration, there is a considerable number of water− water H-bonds present. Hence the complete reorientation relaxation that involves breaking and reforming of H-bonds via water molecules in special H-bonded configurations, in which the hydroxyl group forms a weak, bifurcated H-bond to two other water molecules, will be fast due to abundance in accessing the new H-bond acceptor.30 As more and more TMG is added, the number of TMG−water H-bonds increases. As such, the approach of new water partnera key step in H-bond exchange mechanismwill be hindered by the excluded volume of the neighboring hydrophobic group of TMG similar to TMAO.31 Accordingly, the water reorientation time (τrot) increases with increasing concentration of TMG (Figure 4B). A similar conclusion can also be drawn from fitting the anisotropy decay of HN3 in solutions of TMG (inset of Figure 4B). Sorbitol is larger than TMG and does not possess the bulky trimethyl group instead has six hydroxyl groups. The ability of sorbitol to influence water molecules beyond its first hydration shell is well reflected in the τrot. For the same concentration of osmolyte, the increase in τrot compared to bulk water value is higher for sorbitol than that of TMG (Figure 4B), suggesting that sorbitol-induced perturbation to the orientational dynamics of water is more diffusive and long-ranged in comparison to TMG, as also confirmed by MD simulation and FTIR studies. Similar increase in τrot of HN3 (inset of Figure 4B) is observed with increasing sorbitol concentration, but with an offset value in the anisotropy decay that arises from the NNNsorbitol H-bonding (see Figure S12 of the Supporting Information, where both the amplitude (A) of τrot and the offset (B) are plotted as a function of sorbitol concentrations). However, the contribution from the hydroxyl groups of sorbitol remains small (
