Thermophoresis of a Colloidal Rod: Contributions of Charge and Grafted Polymers.

In this study, we investigated the thermodiffusion behavior of a colloidal model system as a function of the Debye length, λDH, which is controlled by the ionic strength. Our system consists of an fd-virus grafted with poly(ethylene glycol) (PEG) with a molecular mass of 5000 g mol-1. The results are compared with recent measurements on a bare fd-virus and with results of PEG. The diffusion coefficients of both viruses are comparable and increase with the increasing Debye length. The thermal diffusion coefficient, DT, of the bare virus increases strongly with the Debye length, whereas DT of the grafted fd-virus shows only a very weak increase. The Debye length dependence of both systems can be described with an expression derived for charged rods using the surface charge density and an offset of DT as adjustable parameters. It turns out that the ratio of the determined surface charges is inverse to the ratio of the surfaces of the two systems, which means that the total charge remains almost constant. The determined offset of the grafted fd-virus describing the chemical contributions is the sum of DT of PEG and the offset of the bare fd-virus. At high λDH, corresponding to the low ionic strength, the ST values of both colloidal model systems approach each other. This implies a contribution from the polymer layer, which is strong at short λDH and fades out for the longer Debye lengths, when the electric double layer reaches further than the polymer chains and therefore dominates interactions with the surrounding water.

Thermodiffusion and hydrolysis of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

Presently, microfluidic traps are designed mimicking the environment of hydrothermal pores, where a combination of thermophoresis and convection leads to accumulation so that high concentrations of organic matter can be reached. Such a setup is interesting in the context of the origin of life to observe accumulation and possible further synthesis of small organic molecules or prebiotic molecules such as nucleotides or RNA-fragments, but could also be used to replicate DNA-strands. The addition of coupling agents for the activation of carboxyl or phosphate groups such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and EDC-hydrochloride (EDC-HCl) is necessary in order to speed up the process. This work characterizes the thermophoretic properties of EDC and EDC-HCl needed to optimize the design of the traps. At p H 4-6 spontaneous hydrolysis of EDC is observed, which also leads to a neutralisation of the p H. In order to evaluate the thermodiffusion measurements the rate constants were measured at 23 and [Formula: see text] C and the activation energy of the hydrolysis calculated.

Correlation between thermophoretic behavior and hydrophilicity for various alcohols⋆.

Recent experiments for various amides and sugars showed a clear correlation of the temperature dependence of the Soret coefficient with the hydrophilicity, quantitatively described by the logarithm of the 1-octanol/water partition coefficient log P . This coefficient is a measure for the hydrophilicity/hydrophobicity balance of a solute and is often used to model the transport of a compound in the environment or to screen for potential pharmaceutical compounds. In order to validate whether this concept works also for other water soluble molecules we investigated systematically the thermophoresis of mono- and polyhydric alcohols. As experimental method we use a holographic grating technique called infrared Thermal Diffusion Forced Rayleigh Scattering (IR-TDFRS). Experiments showed that the temperature dependence of the Soret coefficient of polyhydric alcohols also correlates with log P and lies on the same master plot as amides and sugars.

Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases.

Formamide is one of the important compounds from which prebiotic molecules can be synthesized, provided that its concentration is sufficiently high. For nucleotides and short DNA strands, it has been shown that a high degree of accumulation in hydrothermal pores occurs, so that temperature gradients might play a role in the origin of life [Baaske P, et al. (2007)Proc Natl Acad Sci USA104(22):9346-9351]. We show that the same combination of thermophoresis and convection in hydrothermal pores leads to accumulation of formamide up to concentrations where nucleobases are formed. The thermophoretic properties of aqueous formamide solutions are studied by means of Infrared Thermal Diffusion Forced Rayleigh Scattering. These data are used in numerical finite element calculations in hydrothermal pores for various initial concentrations, ambient temperatures, and pore sizes. The high degree of formamide accumulation is due to an unusual temperature and concentration dependence of the thermophoretic behavior of formamide. The accumulation fold in part of the pores increases strongly with increasing aspect ratio of the pores, and saturates to highly concentrated aqueous formamide solutions of ∼85 wt% at large aspect ratios. Time-dependent studies show that these high concentrations are reached after 45-90 d, starting with an initial formamide weight fraction of[Formula: see text]wt % that is typical for concentrations in shallow lakes on early Earth.

Unravelling the hydrophobicity of urea in water using thermodiffusion: implications for protein denaturation.

Urea is widely used as a protein denaturant in aqueous solutions. Experimental and computer simulation studies have shown that it dissolves in water almost ideally at high concentrations, introducing little disruption in the water hydrogen bonded structure. However, at concentrations of the order of 5 M or higher, urea induces denaturation in a wide range of proteins. The origin of this behaviour is not completely understood, but it is believed to stem from a balance between urea-protein and urea-water interactions, with urea becoming possibly hydrophobic at a specific concentration range. The small changes observed in the water structure make it difficult to connect the denaturation effects to the solvation properties. Here we show that the exquisite sensitivity of thermodiffusion to solute-water interactions allows the identification of the onset of hydrophobicity of urea-water mixtures. The hydrophobic behaviour is reflected in a sign reversal of the temperature dependent slope of the Soret coefficient, which is observed, both in experiments and non-equilibrium computer simulations at ∼5 M concentration of urea in water. This concentration regime corresponds to the one where abrupt changes in the denaturation of proteins are commonly observed. We show that the onset of hydrophobicity is intrinsically connected to the urea-water interactions. Our results allow us to identify correlations between the Soret coefficient and the partition coefficient, log P, hence establishing the thermodiffusion technique as a powerful approach to study hydrophobicity.

Peptide model systems: Correlation between thermophilicity and hydrophilicity.

In recent years, the response of biomolecules to a temperature gradient has been utilized to monitor reactions of biomolecules, but the underlying mechanism is not well understood due to the complexity of the multicomponent system. To identify some underlying principles, we investigate the thermal diffusion of small amide molecules in water systematically. We re-analyze previous measurements of urea and formamide and compare the results with acetamide, N-methylformamide, and N,N-dimethylformamide, amides with a lower hydrophilicity. It turns out that less hydrophilic substances do not show the typical temperature dependence of water soluble macromolecules. Analyzing temperature and concentration dependent measurements using an empirical expression originally derived for nonpolar mixtures, we find that the so-called isotope contribution depends strongly on the hydrophilicity of the solute. This can be qualitatively understood by comparing with molecular dynamic simulations of Lennard-Jones fluids. The hydrophobic/hydrophilic balance also influences the structure in the fluid and with that the thermal expansion coefficient, which correlates with the thermal diffusion coefficient. Furthermore, we observe a clear correlation of the temperature and concentration dependence of the Soret coefficient with the hydrophilicity, which can be quantitatively described by the partition coefficient log P.

Role of Hydrogen Bonding of Cyclodextrin-Drug Complexes Probed by Thermodiffusion.

Temperature gradient-induced migration of biomolecules, known as thermophoresis or thermodiffusion, changes upon ligand binding. In recent years, this effect has been used to determine protein-ligand binding constants. The mechanism through which thermodiffusive properties change when complexes are formed, however, is not understood. An important contribution to thermodiffusive properties originates from the thermal response of hydrogen bonds. Because there is a considerable difference between the degree of solvation of the protein-ligand complex and its isolated components, ligand-binding is accompanied by a significant change in hydration. The aim of the present work is therefore to investigate the role played by hydrogen bonding on the change in thermodiffusive behavior upon ligand-binding. As a model system, we use cyclodextrins (CDs) and acetylsalicylic acid (ASA), where quite a significant change in hydration is expected and where no conformational changes occur when a CD/ASA complex is formed in aqueous solution. Thermophoresis was investigated in the temperature range of 10-50 °C by infrared thermal diffusion forced Rayleigh scattering. Nuclear magnetic resonance measurements were performed at 25 °C to obtain information about the structure of the complexes. All CD/ASA complexes show a stronger affinity toward regions of lower temperature compared to the free CDs. We found that the temperature sensitivity of thermophoresis correlates with the 1-octanol/water partition coefficient. This observation not only establishes the relation between thermodiffusion and degree of hydrogen bonding but also opens the possibility to relate thermodiffusive properties of complexes to their partition coefficient, which cannot be determined otherwise. This concept is especially interesting for protein-ligand complexes where the protein undergoes a conformational change, different from the CD/ASA complexes, giving rise to additional changes in their hydrophilicity.

Thermophoresis of cyclic oligosaccharides in polar solvents.

Cyclodextrins are cyclic oligosaccharides which are interesting as drug delivery systems, because they can be used as containers for pharmaceutical substances. We studied the Ludwig-Soret effect of [Formula: see text]-, [Formula: see text]-, [Formula: see text]- and methyl-[Formula: see text]-cyclodextrin in water and formamide by infrared thermal diffusion forced Rayleigh scattering (IR-TDFRS). In water the Soret coefficient, S T, of [Formula: see text]-, [Formula: see text]- and [Formula: see text]-cyclodextrin increases with increasing temperature and shows a sign change from negative to positive around T = 35 ° C, while S T of methyl-[Formula: see text]-cyclodextrin is positive in the entire investigated temperature. In formamide S T-values of all cyclodextrins coincide and show a slight decrease with temperature. We discuss the obtained results and relate the S T-values to the different hydrogen bonding capabilities of the cyclodextrins and the used solvents. It turns out that the change of S T with temperature correlates with the partition coefficient, logP, which indicates that more hydrophilic substances show a more pronounced temperature sensitivity of S T. Additionally we obtained a surprising result measuring the refractive index contrast factor with temperature, [Formula: see text] of cyclodextrins in formamide, which might be explained by a complex formation between cyclodextrins and formamide.

Thermophoresis: The Case of Streptavidin and Biotin.

Thermophoretic behavior of a free protein changes upon ligand binding and gives access to information on the binding constants. The Soret effect has also been proven to be a promising tool to gain information on the hydration layer, as the temperature dependence of the thermodiffusion behavior is sensitive to solute-solvent interactions. In this work, we perform systematic thermophoretic measurements of the protein streptavidin (STV) and of the complex STV with biotin (B) using thermal diffusion forced Rayleigh scattering (TDFRS). Our experiments show that the temperature sensitivity of the Soret coefficient is reduced for the complex compared to the free protein. We discuss our data in comparison with recent quasi-elastic neutron scattering (QENS) measurements. As the QENS measurement has been performed in heavy water, we perform additional measurements in water/heavy water mixtures. Finally, we also elucidate the challenges arising from the quantiative thermophoretic study of complex multicomponent systems such as protein solutions.

Strong Adverse Contribution of Conformational Dynamics to Streptavidin-Biotin Binding.

Molecular dynamics plays an important role for the biological function of proteins. For protein ligand interactions, changes of conformational entropy of protein and hydration layer are relevant for the binding process. Quasielastic neutron scattering (QENS) was used to investigate differences in protein dynamics and conformational entropy of ligand-bound and ligand-free streptavidin. Protein dynamics were probed both on the fast picosecond time scale using neutron time-of-flight spectroscopy and on the slower nanosecond time scale using high-resolution neutron backscattering spectroscopy. We found the internal equilibrium motions of streptavidin and the corresponding mean square displacements (MSDs) to be greatly reduced upon biotin binding. On the basis of the observed MSDs, we calculated the difference of conformational entropy ΔSconf of the protein component between ligand-bound and ligand-free streptavidin. The rather large negative ΔSconf value (-2 kJ mol-1 K-1 on the nanosecond time scale) obtained for the streptavidin tetramer seems to be counterintuitive, given the exceptionally high affinity of streptavidin-biotin binding. Literature data on the total entropy change ΔS observed upon biotin binding to streptavidin, which includes contributions from both the protein and the hydration water, suggest partial compensation of the unfavorable ΔSconf by a large positive entropy gain of the surrounding hydration layer and water molecules that are displaced during ligand binding.

Thermodiffusion of Monovalent Organic Salts in Water.

The ionic Soret effect induced by temperature gradients is investigated in organic electrolytes (tetramethylammonium and tetrabutylammonium hydroxides) dispersed in water using a holographic grating experiment. We report the influences of temperature and salt concentrations on the Soret, diffusion, and thermal diffusion coefficients. Experimental results to the thermal diffusion coefficient are compared with a theoretical description for thermodiffusion of Brownian particles in liquids based in the thermal expansion of the liquid solution. It is observed that the obtained thermal diffusion coefficients for the organic electrolytes present a similar temperature dependence as the theoretical prediction. Comparing the experimental results for the organic and common inorganic salts it is proposed an additional physical mechanism as the cause to the different thermal diffusion coefficients in both types of salt. We propose that the temperature dependence of hydration free energy gives rise to a force term that also leads to ion migration in a temperature gradient. We describe the thermal diffusion results as a competition between thermal expansion and hydration effects. The specific structure each type of ion cause in water molecules is considered in the heat of transport theory to describe thermal diffusion of electrolytes. A qualitative agreement is seen between our results and the classical heat of transport theory.