Weakly Hydrated Solute of Mixed Hydrophobic-Hydrophilic Nature.

Infrared (IR) spectroscopy is a commonly used and invaluable tool in studies of solvation phenomena in aqueous solutions. Concurrently, density functional theory calculations and ab initio molecular dynamics simulations deliver the solvation shell picture at the molecular detail level. The mentioned techniques allowed us to gain insights into the structure and energy of the hydrogen bonding network of water molecules around methylsulfonylmethane (MSM). In the hydration sphere of MSM, there are two types of populations of water molecules: a significant share of water molecules weakly bonded to the sulfone group and a smaller share of water molecules strongly bonded to each other around the methyl groups of MSM. The very weak hydrogen bond of water molecules with the hydrophilic group causes the extended network of water hydrogen bonds to be not "anchored" on the sulfone group, and consequently, the MSM hydration shell is labile.

Mechanism of Osmolyte Stabilization-Destabilization of Proteins: Experimental Evidence.

In this work, we investigated the influence of stabilizing (N,N,N-trimethylglycine) and destabilizing (urea) osmolytes on the hydration spheres of biomacromolecules in folded forms (trpzip-1 peptide and hen egg white lysozyme─hewl) and unfolded protein models (glycine─GLY and N-methylglycine─NMG) by means of infrared spectroscopy. GLY and NMG were clearly limited as minimal models for unfolded proteins and should be treated with caution. We isolated the spectral share of water changed simultaneously by the biomacromolecule/model molecule and the osmolyte, which allowed us to provide unambiguous experimental arguments for the mechanism of stabilization/destabilization of proteins by osmolytes. In the case of both types of osmolytes, the decisive factor determining the equilibrium folded/unfolded state of protein was the enthalpy effect exerted on the hydration spheres of proteins in both forms. In the case of stabilizing osmolytes, enthalpy was also favored by entropy, as the unfolded state of a protein was more entropically destabilized than the folded state.

Hydration of Simple Model Peptides in Aqueous Osmolyte Solutions.

The biology and chemistry of proteins and peptides are inextricably linked with water as the solvent. The reason for the high stability of some proteins or uncontrolled aggregation of others may be hidden in the properties of their hydration water. In this study, we investigated the effect of stabilizing osmolyte-TMAO (trimethylamine N-oxide) and destabilizing osmolyte-urea on hydration shells of two short peptides, NAGMA (N-acetyl-glycine-methylamide) and diglycine, by means of FTIR spectroscopy and molecular dynamics simulations. We isolated the spectroscopic share of water molecules that are simultaneously under the influence of peptide and osmolyte and determined the structural and energetic properties of these water molecules. Our experimental and computational results revealed that the changes in the structure of water around peptides, caused by the presence of stabilizing or destabilizing osmolyte, are significantly different for both NAGMA and diglycine. The main factor determining the influence of osmolytes on peptides is the structural-energetic similarity of their hydration spheres. We showed that the chosen peptides can serve as models for various fragments of the protein surface: NAGMA for the protein backbone and diglycine for the protein surface with polar side chains.

Unique agreement of experimental and computational infrared spectroscopy: a case study of lithium bromide solvation in an important electrochemical solvent.

Infrared (IR) spectroscopy is a widely used and invaluable tool in the studies of solvation phenomena in electrolyte solutions. Using state-of-the-art chemometric analysis of a spectral series measured in a concentration-dependent manner, the spectrum of the solute-affected solvent can be extracted, providing a detailed view of the structural and energetic states of the solvent molecules influenced by the solute. Concurrently, ab initio molecular dynamics (AIMD) simulations provide the solvation shell picture at an atomistic detail level and allow for a consistent decomposition of the theoretical IR spectrum in terms of distance-dependent contributions of the solvent molecules. Here, we show for the first time how the chemometric techniques designed with the analysis of experimental data in mind can be harnessed to extract corresponding information from the computed IR spectra for mutual benefit, but without any mutual input. The wide applicability of this two-track approach is demonstrated using lithium bromide solvation in γ-butyrolactone (GBL) as a showcase. GBL is a cyclic ester with extensive applications as a solvent in electrochemistry and we are particularly motivated by its usefulness in the rechargeable cell industry which justifies further studies of lithium cation solvation in GBL. The combination of experiment and simulations firmly asserts the strong solvent structuring character of Li+ and a comparatively weak influence exerted on the solvent by Br-.

General Mechanism of Osmolytes' Influence on Protein Stability Irrespective of the Type of Osmolyte Cosolvent.

The stability of proteins in an aqueous solution can be modified by the presence of osmolytes. The hydration sphere of stabilizing osmolytes is strikingly similar to the enhanced hydration sphere of a protein. This similarity leads to an increase in the protein stability. Moreover, the hydration sphere of destabilizing osmolytes is significantly different. These solutes generate in their surroundings so-called "structurally different water". The addition of such osmolytes causes "dissolution" of the specific protein hydration sphere and destabilizes its folded form. No relationship is seen between the stabilizing/destabilizing properties of osmolytes and their structure-making/-breaking influence on water. Furthermore, their accumulation at the protein surface or their exclusion does not determine the osmolytes' effect on protein stability. An explanation to the osmolytes' stabilizing/destabilizing influence originates in the similarity of water properties in osmolytes and protein solutions. The spectral infrared characteristic of water in an osmolyte solution allowed us to develop practical criteria for classifying solutes as stabilizing or destabilizing agents.

Molecular basis of the osmolyte effect on protein stability: a lesson from the mechanical unfolding of lysozyme.

Osmolytes are a class of small organic molecules that shift the protein folding equilibrium. For this reason, they are accumulated by organisms under environmental stress and find applications in biotechnology where proteins need to be stabilized or dissolved. However, despite years of research, debate continues over the exact mechanisms underpinning the stabilizing and denaturing effect of osmolytes. Here, we simulated the mechanical denaturation of lysozyme in different solvent conditions to study the molecular mechanism by which two biologically relevant osmolytes, denaturing (urea) and stabilizing (betaine), affect the folding equilibrium. We found that urea interacts favorably with all types of residues via both hydrogen bonds and dispersion forces, and therefore accumulates in a diffuse solvation shell around the protein. This not only provides an enthalpic stabilization of the unfolded state, but also weakens the hydrophobic effect, as hydrophobic forces promote the association of urea with nonpolar residues, facilitating the unfolding. In contrast, we observed that betaine is excluded from the protein backbone and nonpolar side chains, but is accumulated near the basic residues, yielding a nonuniform distribution of betaine molecules at the protein surface. Spatially resolved solvent-protein interaction energies further suggested that betaine behaves in a ligand- rather than solvent-like manner and its exclusion from the protein surface arises mostly from the scarcity of favorable binding sites. Finally, we found that, in the presence of betaine, the reduced ability of water molecules to solvate the protein results in an additional enthalpic contribution to the betaine-induced stabilization.

Protein thermal stabilization in aqueous solutions of osmolytes.

Proteins' thermal stabilization is a significant problem in various biomedical, biotechnological, and technological applications. We investigated thermal stability of hen egg white lysozyme in aqueous solutions of the following stabilizing osmolytes: Glycine (GLY), N-methylglycine (NMG), N,N-dimethylglycine (DMG), N,N,N-trimethylglycine (TMG), and trimethyl-N-oxide (TMAO). Results of CD-UV spectroscopic investigation were compared with FTIR hydration studies' results. Selected osmolytes increased lysozyme's thermal stability in the following order: Gly>NMG>TMAO≈DMG>TMG. Theoretical calculations (DFT) showed clearly that osmolytes' amino group protons and water molecules interacting with them played a distinctive role in protein thermal stabilization. The results brought us a step closer to the exact mechanism of protein stabilization by osmolytes.

Hydration of amino acids: FTIR spectra and molecular dynamics studies.

The hydration of selected amino acids, alanine, glycine, proline, valine, isoleucine and phenylalanine, has been studied in aqueous solutions by means of FTIR spectra of HDO isotopically diluted in H2O. The difference spectra procedure and the chemometric method have been applied to remove the contribution of bulk water and thus to separate the spectra of solute-affected HDO. To support interpretation of obtained spectral results, molecular dynamics simulations of amino acids were performed. The structural-energetic characteristic of these solute-affected water molecules shows that, on average, water affected by amino acids forms stronger and shorter H-bonds than those in pure water. Differences in the influence of amino acids on water structure have been noticed. The effect of the hydrophobic side chain of an amino acid on the solvent interactions seems to be enhanced because of the specific cooperative coupling of water strong H-bond chain, connecting the carboxyl and amino groups, with the clathrate-like H-bond network surrounding the hydrocarbon side chain. The parameter derived from the spectral data, which corresponds to the contributions of the population of weak hydrogen bonds of water molecules which have been substituted by the stronger ones in the hydration sphere of amino acids, correlated well with the amino acid hydrophobicity indexes.

Influence of osmolytes on protein and water structure: a step to understanding the mechanism of protein stabilization.

Results concerning the thermostability of hen egg white lysozyme in aqueous solutions with stabilizing osmolytes, trimethylamine-N-oxide (TMAO), glycine (Gly), and its N-methyl derivatives, N-methylglycine (NMG), N,N-dimethylglycine (DMG), and N,N,N-trimethylglycine (betaine, TMG), have been presented. The combination of spectroscopic (IR) and calorimetric (DSC) data allowed us to establish a link between osmolytes' influence on water structure and their ability to thermally stabilize protein molecule. Structural and energetic characteristics of stabilizing osmolytes' and lysozyme's hydration water appear to be very similar. The osmolytes increase lysozyme stabilization in the order bulk water < TMAO < TMG < Gly < DMG < NMG, which is consistent with the order corresponding to the value of the most probable oxygen-oxygen distance of water molecules affected by osmolytes in their surrounding. Obtained results verified the hypothesis concerning the role of water molecules in protein stabilization, explained the osmophobic effect, and finally helped to bring us nearer to the exact mechanism of protein stabilization by osmolytes.

Chemometric method of spectra analysis leading to isolation of lysozyme and CtDNA spectra affected by osmolytes.

In this paper we present a chemometric method of analysis leading to isolation of Fourier transform infrared (FT-IR) spectra of biomacromolecules (HEW lysozyme, ctDNA) affected by osmolytes (trimethylamine-N-oxide and N,N,N-trimethylglycine, respectively) in aqueous solutions. The method is based on the difference spectra method primarily used to characterize the structure of solvent affected by solute. The cyclical usage of factor analysis allows precise information to be obtained on the shape of "affected spectra" of analyzed biomacromolecules. "Affected spectra" of selected biomacromolecules give valuable information on their structure in the presence of the osmolytes in solution, as well as on the level of perturbation in dependence of osmolyte concentration. The method also gives a possibility of insight into the mechanism of interaction in presented types of systems. It can be easily adapted to various chemical and biochemical problems where vibrational or ultraviolet-visible (UV-Vis) spectroscopy is used.

Characteristics of hydration water around hen egg lysozyme as the protein model in aqueous solution. FTIR spectroscopy and molecular dynamics simulation.

In this paper, the hydration of a model protein--hen egg white lysozyme in aqueous solution has been presented. The leading method used was FTIR spectroscopy with an application of a technique of semi-heavy water (HDO) isotope dilution. Analysis of spectra of HDO isotopically diluted in water solution of lysozyme allowed us to isolate HDO spectra affected by lysozyme, and thus to characterise the energetic state of water molecules and their arrangement around protein molecules. The number of water molecules and the shape of the affected HDO spectrum were obtained using a classical and a chemometric method. This shape showed that the HDO spectrum affected by lysozyme may be presented as a superposition of two spectra corresponding to HDO affected by N-methylacetamide and the carboxylate anion (of the formic acid). Moreover, based on the difference in intermolecular distances distribution of water molecules (obtained from spectral data), we demonstrated that the lysozyme molecule causes a decrease in population of weak hydrogen bonds, and concurrently increases the probability of an occurrence of short hydrogen bonds in water affected by lysozyme. This conclusion was also confirmed by the molecular dynamics (MD) simulation.

Application of the tetraphenylphosphonium tetraphenylborate (TPTB) assumption to the thermodynamic properties of solvated ions in dimethylsulfoxide.

The extra-thermodynamic tetraphenylphosphonium tetraphenylborate assumption has been tested for dimethylsulfoxide using ATR FTIR spectroscopy. Solute-affected DMSO spectra show that, contrary to the TPTB assumption, the charge density on BPh(4)(-) and Ph(4)P(+) ions is sufficiently high to influence the DMSO molecules orientation with respect to the cation and to the anion. Apparently, the Ph(4)P(+) cation does not affect the structure of DMSO whereas the BPh(4)(-) anion clearly breaks it up. Our results indicate that the TPTB extra-thermodynamic assumption is not a sound basis for splitting thermodynamic values obtained for DMSO solutions into ionic contributions.

Intramolecular interactions in crystals of tris(2,6-diisopropylphenoxy)silanethiol and its sodium salts.

Hydrolytically stable silanethiol tris(2,6-diisopropylphenoxy)silanethiol (TDST) has been synthesized and reacted with sodium metal. In solid state TDST exhibits π-interactions between the S-H unit and the π-system of the arene, replaced by cation-π interactions in its sodium salts. The interactions are documented by crystal structures and FT-IR spectroscopy.

Hydration of simple carboxylic acids from infrared spectra of HDO and theoretical calculations.

The hydration of carboxylic acids in dilute aqueous solutions is important for our understanding of their functioning in the biochemical context. Here we apply vibrational spectra of HDO isotopically diluted in H(2)O to study this phenomenon, using the difference spectra method for analysis and interpretation of the results. The spectra of HDO affected by formic, acetic, and propionic acid display characteristic component bands, significantly red-shifted from the bulk HDO band position. The appearance of these component bands is linked with isotopic substitution on the carboxylic acid molecule, which forms a short and strong hydrogen bond with a water molecule. Additionally, a charge separation due to the proton transfer in the neutral form of the complex leading to a contact ion pair formation may be inferred from the affected HDO spectra. Apart from the contraction of the principal acid-water hydrogen bond, it results in other major structural changes in the hydration shell, as revealed by density functional theory (DFT) calculations of optimal geometries of aqueous clusters of the studied acids.

Fourier transform infrared spectroscopic and theoretical study of water interactions with glycine and its N-methylated derivatives.

In this study we attempt to explain the molecular aspects of amino acids' hydration. Glycine and its N-methylated derivatives: N-methylglycine, N,N-dimethylglycine, and N,N,N-trimethylglycine were used as model solutes in aqueous solution, applying FT-IR spectroscopy as the experimental method. The quantitative version of the difference spectra method enabled us to obtain the solute-affected HDO spectra as probes of influenced water. The spectral results were confronted with density functional theory calculated structures of small hydration complexes of the solutes using the polarizable continuum model. It appears that the hydration of amino acids in the zwitterionic form can be understood allowing a synchronized fluctuation of hydrogen bonding between the solute and the water molecules. This effect is caused by a noncooperative interaction of water molecules with electrophilic groups of amino acid and by intramolecular hydrogen bond, allowing proton transfer from the carboxylic to the amine group, accomplishing by the chain of two to four water molecules. As a result, an instantaneous water-induced asymmetry of the carboxylate and the amino group of amino acid molecule is observed and recorded as HDO band splitting. Water molecules interacting with the carboxylate group give component bands at 2543 ± 11 and 2467 ± 15 cm(-1), whereas water molecules interacting with protons of the amine group give rise to the bands at 2611 ± 15 and 2413 ± 12 cm(-1). These hydration effects have not been recognized before and there are reasons to expect their validity for other amino acids.

An effective method for studying intermolecular interactions in binary liquids with hydrogen bonds; FTIR spectra and ab initio calculations in the N-methylformamide-methanol system.

Molecular complexes in methanol (MeOH)-N-methylformamide (NMF) mixtures were studied based on their FTIR-ATR spectra, to which two methods of analysis were applied: factor analysis and a quantitative version of the difference-spectra method. The mean composition of a complex between NMF and MeOH molecules over the whole range of mixture compositions was determined. Absorbing species differentiated with regard to the interaction energies of the carbonyl oxygen with methanol molecules were recognized in both compositional regions with a marked excess of one component. Possible structures for complexes of various stoichiometries were optimized by ab initio calculations in the gas phase and both liquid NMF and MeOH using the polarizable continuum model (PCM). Thermodynamic functions calculated for the optimized structures were used to find the most stable structure for each stoichiometry. Individuals distinguished by the spectral analysis were assigned to the complexes of definite composition, and a linear correlation between the positions of the carbonyl group absorption and the total interaction energies of the complexes was found. The results of the spectral analysis of the NMF-MeOH mixtures were compared to those we obtained previously for similar binary systems, i.e., mixtures of methanol and formamide (FA) or N,N-dimethylformamide (DMF). It was shown that the factor analysis applied to the infrared spectra is an effective method for distinguishing molecular complexes with different polarizations of component molecules and allows for the detection of even weak intermolecular interactions and low-concentration species. Combined with the difference-spectra method, factor analysis provides a comprehensive picture of intermolecular interactions in binary mixtures.

Preclusion of irreversible destruction of Dr adhesin structures by a high activation barrier for the unfolding stage of the fimbrial DraE subunit.

Dr fimbriae of uropathogenic Eschericha coli strains are an example of surface-located adhesive structures assembled via the chaperone-usher pathway. These structures are crucial for specific attachment of bacteria to host receptors. Dr fimbriae are linear associates of DraE proteins, the structure of which is determined by a donor strand complementation between the consecutive subunits. The biogenesis of these structures is dependent on a function of the specific periplasmic chaperone and outer membrane usher proteins. In a consequence of these structural and assembly properties the potential unfolding of a single subunit in a linear associate would cause a destruction of fimbrial adhesion function. This correlates with the observed high resistance of fimbrial structures for denaturation. In this paper we show that the mechanism of thermal denaturation of DraE-sc protein is well described by an irreversible two-state model which is the reduced form of a Lumry-Eyring protein denaturation model. In theory of this model the observed stability of DraE-sc protein is determined by the high activation barrier for the unfolding stage N-->U. The microcalorimetry experiments permit to determine kinetic parameters of the DraE-sc unfolding process: energy of activation of 463.5 +/- 20.8 kJ.mol(-1) and rate constant of order 10(-17) s(-1). This corresponds to the dissociation/unfolding half-life of Dr fimbriae of 10(8) years at 25 degrees C. The FT-IR experiments show that the high stability of DraE is determined by the cooperative rigid protein core. The presented mechanism of kinetic stability of Dr fimbriae is probably universal to adhesive structures of the chaperone-usher type.

Effects of urea and trimethylamine-N-oxide on the properties of water and the secondary structure of hen egg white lysozyme.

The influence of urea and trimethylamine-N-oxide (TMAO) on the structure of water and secondary structure of hen egg white lysozyme (HEWL) has been investigated. The hydration of these osmolytes was studied in aqueous solutions by means of FTIR spectra of HDO isotopically diluted in H(2)O. The difference spectra procedure was applied to remove the contribution of bulk water and thus to separate the spectra of solute-affected HDO. The structural-energetic characteristic of these solute-affected water molecules shows that, on average, water affected by TMAO forms stronger H-bonds and is more ordered than pure water. In the case of urea, the H-bonds are very similar to those in pure water. To facilitate the interpretation of the obtained spectral results, calorimetric measurements, DFT calculations, and molecular dynamics (MD) simulations of aqueous osmolyte clusters were performed. All of these results confirmed that the interactions of TMAO with water molecules are much stronger than those of urea with water. Additional ATR FTIR measurements were performed to characterize the influence of the examined osmolytes on the secondary structure of HEW lysozyme. The type of interactions (direct or indirect) was determined, based on the second derivatives of ATR protein spectra record during an increase in the osmolyte concentration. The changes in the amide I band shape caused by urea or TMAO were found to correlate quite well with changes in the water structure around these osmolytes.

Systematic study of hydration patterns of phosphoric(V) acid and its mono-, di-, and tripotassium salts in aqueous solution.

Fourier transform infrared (FTIR) spectroscopy of the OD band of HDO molecules has been applied to perform a systematic study of various phosphate forms in the order of decreasing protonation: H3PO4, KH2PO4, K2HPO4, K3PO4. HDO isotopically diluted in H2O has been prepared by adding adequate amounts of D2O to aqueous solutions in ordinary water. The difference spectra procedure has been applied to remove the contribution of bulk water and thus to separate the spectra of solute-affected HDO. The position at maximum of the principal anion-affected HDO band for potassium phosphates moves in the order KH2PO4 (2478 cm(-1))>K2HPO4 (2363 cm(-1))>K3PO4 (2301 cm(-1)), that is, decreases with increasing solute basicity and charge. The number of moles of water affected by one mole of solute (N) equals 11.0, 13.8 and 16.2, respectively. Phosphoric acid affects statistically 13.9 water molecules and appears to be a "structure making" solute in water. The isotopic substitution with deuterium occurs also on the phosphate anions and phosphoric acid. The thus formed P-O-D groups interact with water molecules via strong hydrogen bonds and the relative strength of this interaction increases with increasing solute acidity. The plausible assignments of OD bands of HDO have been confirmed by calculating equilibrium structures of small aqueous clusters of the studied individual utilizing density functional theory. Further interpretation of the energetic and structural properties of hydrating water is enabled by calculating intermolecular interaction energy of water and probability distributions for interatomic oxygen-oxygen distance.