Deciphering the guanidinium cation: Insights into thermal diffusion.

Thermophoresis, or thermodiffusion, is becoming a more popular method for investigating the interactions between proteins and ligands due to its high sensitivity to the interactions between solutes and water. Despite its growing use, the intricate mechanisms behind thermodiffusion remain unclear. This gap in knowledge stems from the complexities of thermodiffusion in solvents that have specific interactions as well as the intricate nature of systems that include many components with both non-ionic and ionic groups. To deepen our understanding, we reduce complexity by conducting systematic studies on aqueous salt solutions. In this work, we focused on how guanidinium salt solutions behave in a temperature gradient, using thermal diffusion forced Rayleigh scattering experiments at temperatures ranging from 15 to 35 °C. We looked at the thermodiffusive behavior of four guanidinium salts (thiocyanate, iodide, chloride, and carbonate) in solutions with concentrations ranging from 1 to 3 mol/kg. The guanidinium cation is disk-shaped and is characterized by flat hydrophobic surfaces and three amine groups, which enable directional hydrogen bonding along the edges. We compare our results to the behavior of salts with spherical cations, such as sodium, potassium, and lithium. Our discussions are framed around how different salts are solvated, specifically in the context of the Hofmeister series, which ranks ions based on their effects on the solvation of proteins.

Polyelectrolyte-protein synergism: pH-responsive polyelectrolyte/insulin complexes as versatile carriers for targeted protein and drug delivery.

The co-assembly of polyelectrolytes (PE) with proteins offers a promising approach for designing complex structures with customizable morphologies, charge distribution, and stability for targeted cargo delivery. However, the complexity of protein structure limits our ability to predict the properties of the formed nanoparticles, and our goal is to identify the key triggers of the morphological transition in protein/PE complexes and evaluate their ability to encapsulate multivalent ionic drugs. A positively charged PE can assemble with a protein at pH above isoelectric point due to the electrostatic attraction and disassemble at pH below isoelectric point due to the repulsion. The additional hydrophilic block of the polymer should stabilize the particles in solution and enable them to encapsulate a negatively charged drug in the presence of PE excess. We demonstrated that diblock copolymers, poly(ethylene oxide)-block-poly(N,N-dimethylaminoethyl methacrylate) and poly(ethylene oxide)-block-poly(N,N,N-trimethylammonioethyl methacrylate), consisting of a polycation block and a neutral hydrophilic block, reversibly co-assemble with insulin in pH range between 5 and 8. Using small-angle neutron and X-ray scattering (SANS, SAXS), we showed that insulin arrangement within formed particles is controlled by intermolecular electrostatic forces between protein molecules, and can be tuned by varying ionic strength. For the first time, we observed by fluorescence that formed protein/PE complexes with excess of positive charges exhibited potential for encapsulating and controlled release of negatively charged bivalent drugs, protoporphyrin-IX and zinc(II) protoporphyrin-IX, enabling the development of nanocarriers for combination therapies with adjustable charge, stability, internal structure, and size.

Non-monotonic Soret coefficients of aqueous LiCl solutions with varying concentrations.

We investigate the thermodiffusive properties of aqueous solutions of lithium chloride, using thermal diffusion forced Rayleigh scattering in a concentration range of 0.5-2 mole per kg of solvent and a temperature range of 5 to 45 °C. All solutions exhibit non-monotonic variations of the Soret coefficient ST with a concentration exhibiting a minimum at about one mole per kg of solvent. The depth of the minimum decreases with increasing temperature and shifts slightly towards higher concentrations. We compare the experimental data with published data and apply a recent model based on overlapping hydration shells. Additionally, we calculate the ratio of the phenomenological Onsager coefficients using our experimental results and published data to calculate the thermodynamic factor. Simple linear, quadratic and exponential functions can be used to describe this ratio accurately, and together with the thermodynamic factors, the experimental Soret coefficients can be reproduced. The main conclusion from this analysis is that the minimum of the Soret coefficients results from a maximum in the thermodynamic factor, which appears itself at concentrations far below the experimental concentrations. Only after multiplication by the (negative) monotonous Onsager ratio does the minimum move into the experimental concentration window.

Complementary Experimental Methods to Obtain Thermodynamic Parameters of Protein Ligand Systems.

In recent years, thermophoresis has emerged as a promising tool for quantifying biomolecular interactions. The underlying microscopic physical effect is still not understood, but often attributed to changes in the hydration layer once the binding occurs. To gain deeper insight, we investigate whether non-equilibrium coefficients can be related to equilibrium properties. Therefore, we compare thermophoretic data measured by thermal diffusion forced Rayleigh scattering (TDFRS) (which is a non-equilibrium process) with thermodynamic data obtained by isothermal titration calorimetry (ITC) (which is an equilibrium process). As a reference system, we studied the chelation reaction between ethylenediaminetetraacetic acid (EDTA) and calcium chloride (CaCl2) to relate the thermophoretic behavior quantified by the Soret coefficient ST to the Gibb's free energy ΔG determined in the ITC experiment using an expression proposed by Eastman. Finally, we have studied the binding of the protein Bovine Carbonic Anhydrase I (BCA I) to two different benzenesulfonamide derivatives: 4-fluorobenzenesulfonamide (4FBS) and pentafluorobenzenesulfonamide (PFBS). For all three systems, we find that the Gibb's free energies calculated from ST agree with ΔG from the ITC experiment. In addition, we also investigate the influence of fluorescent labeling, which allows measurements in a thermophoretic microfluidic cell. Re-examination of the fluorescently labeled system using ITC showed a strong influence of the dye on the binding behavior.

Overlapping hydration shells in salt solutions causing non-monotonic Soret coefficients with varying concentration.

We investigate the thermodiffusive properties of aqueous solutions of sodium iodide, potassium iodide and lithium iodide, using thermal diffusion forced Rayleigh scattering in a concentration range of 0.5-4 mol kg-1 of solvent, large enough to deal with associated salts, and a temperature range of 15 to 45 °C. All systems exhibit non-monotonic variations of the Soret coefficient ST with concentration, with a minimum at one mol kg-1 of solvent in all three cases. We take this as an indication that the relevant length and energy scales are very similar in all cases. On this basis we develop an intuitive picture in which the relevant objects are the fully hydrated salt molecules, including all water molecules that behave differently from bulk water. Preliminary, somewhat sketchy calculations indicate that indeed Soret coefficients begin to rise beyond concentrations where the fully hydrated particles are randomly close packed. Indications are given as to why the model will fail at large concentrations.

Towards understanding specific ion effects in aqueous media using thermodiffusion.

Specific ion effects play an important role in scientific and technological processes. According to Hofmeister, the influence on the hydrogen bond network depends on the ion and leads to a specific order of the ions. Also thermodiffusion the mass transport caused by a temperature gradient is very sensitive to changes of the hydrogen bond network leading to a ranking according to hydrophilicity of the salt. Hence, we investigate various salt solutions in order to compare with the Hofmeister concept. We have studied three different sodium salts in water as a function of temperature (25-45[Formula: see text]C) and concentration (0.5-5 mol kg[Formula: see text]) using Thermal Diffusion Forced Rayleigh Scattering (TDFRS). The three anions studied, carbonate, acetate and thiocyanate, span the entire range of the Hofmeister series from hydrophilic to hydrophobic. We compare the results with the recent measurements of the corresponding potassium salts to see to what extent the cation changes the thermodiffusion of the salt.

Temperature profile characterization with fluorescence lifetime imaging microscopy in a thermophoretic chip.

This study introduces a thermophoretic lab-on-a-chip device to measure the Soret coefficient. We use resistive heating of a microwire on the chip to induce a temperature gradient, which is measured by fluorescence lifetime imaging microscopy (FLIM). To verify the functionality of the device, we used dyed polystyrene particles with a diameter of 25 nm. A confocal microscope is utilized to monitor the concentration profile of colloidal particles in the temperature field. Based on the measured temperature and concentration differences, we calculate the corresponding Soret coefficient. The same particles have been recently investigated with thermal diffusion forced Rayleigh scattering (TDFRS) and we find that the obtained Soret coefficients agree with literature results. This chip offers a simple way to study the thermophoretic behavior of biological systems in multicomponent buffer solutions quantitatively, which are difficult to study with optical methods solely relying on the refractive index contrast.

Thermodiffusion of aqueous solutions of various potassium salts.

Thermophoresis or thermodiffusion has become an important tool to monitor protein-ligand binding as it is very sensitive to the nature of solute-water interactions. However, the microscopic mechanisms underlying thermodiffusion in protein systems are poorly understood at this time. One reason is the difficulty to separate the effects of the protein system of interest from the effects of buffers that are added to stabilize the proteins. Due to the buffers, typical protein solutions form multicomponent mixtures with several kinds of salt. To achieve a more fundamental understanding of thermodiffusion of proteins, it is therefore necessary to investigate solutions of buffer salts. For this work, the thermodiffusion of aqueous potassium salt solutions has been studied systematically. We use thermal diffusion forced Rayleigh scattering experiments in a temperature range from 15 °C to 45 °C to investigate the thermodiffusive properties of aqueous solutions of five potassium salts: potassium chloride, potassium bromide, potassium thiocyanate, potassium acetate, and potassium carbonate in a molality range between 1 mol/kg and 5 mol/kg. We compare the thermophoretic results with those obtained for non-ionic solutes and discuss the thermophoresis of the salts in the context of ion-specific solvation according to the Hofmeister series.

Thermophoretic Micron-Scale Devices: Practical Approach and Review.

In recent years, there has been increasing interest in the development of micron-scale devices utilizing thermal gradients to manipulate molecules and colloids, and to measure their thermophoretic properties quantitatively. Various devices have been realized, such as on-chip implements, micro-thermogravitational columns and other micron-scale thermophoretic cells. The advantage of the miniaturized devices lies in the reduced sample volume. Often, a direct observation of particles using various microscopic techniques is possible. On the other hand, the small dimensions lead to some technical problems, such as a precise temperature measurement on small length scale with high spatial resolution. In this review, we will focus on the "state of the art" thermophoretic micron-scale devices, covering various aspects such as generating temperature gradients, temperature measurement, and the analysis of the current micron-scale devices. We want to give researchers an orientation for their development of thermophoretic micron-scale devices for biological, chemical, analytical, and medical applications.

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 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.

Impact of Molecular Crowding on Translational Mobility and Conformational Properties of Biological Macromolecules.

Effects of molecular crowding on structural and dynamical properties of biological macromolecules do depend on the concentration of crowding agents but also on the molecular mass and the structural compactness of the crowder molecules. By employing fluorescence correlation spectroscopy (FCS), we investigated the translational mobility of several biological macromolecules ranging from 17 kDa to 2.7 MDa. Polyethylene glycol and Ficoll polymers of different molecular masses were used in buffer solutions to mimic a crowded environment. The reduction in translational mobility of the biological tracer molecules was analyzed as a function of crowder volume fractions and was generally more pronounced in PEG as compared to Ficoll solutions. For several crowding conditions, we observed a molecular sieving effect, in which the diffusion coefficient of larger tracer molecules is reduced to a larger extent than predicted by the Stokes-Einstein relation. By employing a FRET-based biosensor, we also showed that a multiprotein complex is significantly compacted in the presence of macromolecular crowders. Importantly, with respect to sensor in vivo applications, ligand concentration determining sensors would need a crowding specific calibration in order to deliver correct cytosolic ligand concentration.

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.

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.

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.

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.

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.

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.

Influence of temperature and charge effects on thermophoresis of polystyrene beads⋆.

We study the thermodiffusion behavior of spherical polystyrene beads with a diameter of 25 nm by infrared thermal diffusion Forced Rayleigh Scattering (IR-TDFRS). Similar beads were used to investigate the radial dependence of the Soret coefficient by different authors. While Duhr and Braun (Proc. Natl. Acad. Sci. U.S.A. 104, 9346 (2007)) observed a quadratic radial dependence Braibanti et al. (Phys. Rev. Lett. 100, 108303 (2008)) found a linear radial dependence of the Soret coefficient. We demonstrated that special care needs to be taken to obtain reliable thermophoretic data, because the measurements are very sensitive to surface properties. The colloidal particles were characterized by transmission electron microscopy and dynamic light scattering (DLS) experiments were performed. We carried out systematic thermophoretic measurements as a function of temperature, buffer and surfactant concentration. The temperature dependence was analyzed using an empirical formula. To describe the Debye length dependence we used a theoretical model by Dhont. The resulting surface charge density is in agreement with previous literature results. Finally, we analyze the dependence of the Soret coefficient on the concentration of the anionic surfactant sodium dodecyl sulfate (SDS), applying an empirical thermodynamic approach accounting for chemical contributions.