Thermophoresis of biological and biocompatible systems Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakult¨at der Universit¨at zu K¨oln vorgelegt von Doreen Niether aus Eberswalde-Finow K¨oln, 2018 Berichterstatter: Prof. Dr. Annette Schmidt (Gutachter) Prof. Dr. Simone Wiegand Prof. Dr. Guillaume Galliero Tag der mundlichen¨ Prufung:¨ 17.05.2018 i Abstract Thermophoresis, or thermodiffusion, is mass transport driven by a temperature gradient. This work focuses on thermodiffusion in a biological context, where there are two major applications for the effect: accumulation of a component in microfluidic devices through a combination of thermodiffusion and convection, and monitoring of protein binding reac- tions through the sensitivity of thermodiffusion to complex formation. Both applications are investigated, the first as an accumulation process in the context of origin-of-life theories and the second in light of the question what we can learn from the observed changes in thermodiffusion about modifications of the hydration shell upon complex formation. While thermodiffusion in non-polar liquids can be predicted with reasonable accuracy, the descrip- tion of aqueous systems is complicated as their concentration and temperature dependence is often anomalous. The underlying goal of this work is to gain a better understanding of the interactions between components in an aqueous mixture and how they influence thermo- diffusion. We find that the temperature dependence of a solute's thermodiffusion correlates with its hydrophilicity and argue that the temperature sensitivity of hydrogen bonds, which domi- nate the interactions in aqueous solutions, might induce a temperature dependence of the chemical potential. Such a temperature dependence is as of yet not considered in theore- tical descriptions of thermodiffusion. Numerical calculations show that the thermophoretic accumulation process, as of yet only considered for the formation of RNA, can accumulate formamide to high concentrations that would allow the formation of prebiotic molecules. A heuristic model is developed to illuminate the mechanism behind the accumulation. Cyclo- dextrins and streptavidin were investigated as model systems for biological complexes. It is feasible that the exquisite sensitivity of thermodiffusion to interactions with the surrounding solvent allows inferences about changes in the protein's hydration shell upon complex forma- tion. Preliminary measurements on streptavidin-biotin show a decreased hydrophilicity of the complex, which is in qualitative agreement with increased entropy of the hydration shell upon complex formation calculated from calorimetric and neutron scattering experiments. ii Kurzzusammenfassung Thermophorese, oder Thermodiffusion, ist Massentransport, der durch einen Temperaturgra- dienten hervorgerufen wird. Fur¨ Thermodiffusion in einem biologischen Kontext, auf welcher der Fokus dieser Arbeit liegt, gibt es im Wesentlichen zwei Anwendungen: die Akkumulation einer Komponente in mikrofluidischen Systemen durch eine Kombination aus Thermodiffu- sion und Konvektion und die Detektion von Bindungsreaktionen uber¨ die Ver¨anderung in der Thermodiffusion eines Proteins, wenn ein Ligand bindet. Beide Anwendungen werden hier untersucht, Erstere als Anreicherungsprozess im Kontext von Theorien zur Entstehung des Lebens und Letzere im Bezug auf die Frage, inwieweit die beobachteten Anderung¨ der Thermodiffusion Ruckschl¨ usse¨ auf Modifikation der Hydrathulle¨ durch die Komplexbildung zulassen. W¨ahrend die Thermodiffusion von unpolaren Flussigkeiten¨ inzwischen im Wesent- lichen vorhersagbar ist, wird die Beschreibung von w¨assrigen Systemen durch Anomalien der Konzentrations- und Temperaturabh¨angigkeit verkompliziert. Das grundlegende Ziel dieser Arbeit ist deshalb, ein besseres Verst¨andnis der Wechselwirkungen zwischen Komponenten einer w¨assrigen L¨osung und ihres Einflusses auf die Thermodiffusion zu erm¨oglichen. Es konnte gezeigt werden, dass es eine Korrelation zwischen der Temperaturabh¨angigkeit der Thermodiffusion eines gel¨osten Stoffes und seiner Hydrophilie gibt. Dies kann in der Tem- peraturempfindlichkeit von Wasserstoffbruckenbindungen¨ begrundet¨ sein, welche die Wech- selwirkungen in w¨assrigen Systemen dominieren und zu einer Temperaturabh¨angigkeit des chemischen Potentials fuhren¨ k¨onnen. Eine solche Temperaturabh¨angigkeit wird bisher in theoretischen Beschreibungen der Thermodiffusion vernachl¨assigt. Numerische Rechnungen zeigen, dass der thermophoretische Anreicherungsprozess, welcher bislang nur unter dem Gesichtspunkt der Bildung von RNA betrachtet wurde, auch Formamid zu hohen Kon- zentrationen akkumulieren kann, welche die Bildung von pr¨abiotischen Molekulen¨ zulassen wurden.¨ Es wurde ein heuristisches Modell entwickelt, um den Mechanismus der Akkumula- tion zu erl¨autern. Cyclodextrine und Streptavidin wurden als Modellsysteme fur¨ biologische Komplexe untersucht. Die hohe Empfindlichkeit der Thermodiffusion gegenuber¨ den Wech- selwirkungen mit dem umgebenden L¨osungsmittel sollte Aussagen uber¨ Ver¨anderungen in der Hydrathulle¨ des Proteins durch Ligandenbindung zulassen. Vorl¨aufige Untersuchungen an Streptavidin-Biotin zeigen eine reduzierte Hydrophilie des Komplexes, was in qualitativer Ubereinstimmung¨ mit einer Erh¨ohung des entropischen Beitrags der Hydrathulle¨ ist, wie er aus kalorimetrischen und Neutronenstreu-Experimenten berechnet wurde. Contents 1 Introduction1 1.1 Introduction to thermodiffusion............................1 1.1.1 Contributions to the Soret effect.......................2 1.1.2 Thermodynamic thermodiffusion models..................4 1.1.3 Simulations....................................6 1.1.4 Thermodiffusion in aqueous systems.....................7 1.2 Applications........................................8 1.2.1 Thermogravitational columns.........................9 1.2.2 Microscale thermophoresis........................... 10 1.3 Experimental Details.................................. 12 1.3.1 Description of the IR-TDFRS setup..................... 12 1.3.2 Contrast factors................................. 14 1.3.3 Sample preparation............................... 14 1.4 Outline of the thesis................................... 16 2 Accumulation of Formamide in Hydrothermal Pores to Form Prebiotic Nucleobases 19 3 Heuristic Approach to Understanding the Accumulation Process in Hydrothermal Pores 27 iii iv Contents 4 Unravelling the Hydrophobicity of Urea in Water Using Thermodiffusion: Implications for Protein Denaturation 39 5 Thermophoresis of Cyclic Oligosaccharides in Polar Solvents 49 6 Role of Hydrogen Bonding of Cyclodextrin-Drug Complexes Probed by Thermodiffusion 59 7 Thermodiffusion as a Probe of Protein Hydration for Streptavidin and the Streptavidin-Biotin Complex 71 8 Discussion and Conclusion 79 8.1 Discussion......................................... 79 8.1.1 Thermophoretic accumulation......................... 80 8.1.2 Influence of hydrophilicity........................... 81 8.1.3 Thermodiffusion of complexes......................... 87 8.2 Conclusion......................................... 89 8.2.1 Accumulation in a hydrothermal pore.................... 89 8.2.2 Hydrophilicity and the temperature dependence of ST .......... 90 8.2.3 Change of thermodiffusion behaviour upon complex formation..... 91 8.3 Outlook.......................................... 92 Acknowledgement 95 Bibliography 97 Appendix 105 Supporting Information.................................... 105 Declaration of Individual Contribution........................... 150 Erkl¨arung zur Dissertation.................................. 153 Lebenslauf............................................ 154 1 Introduction 1.1 Introduction to thermodiffusion Thermodiffusion is mass transport driven by a temperature gradient. It is also known as thermophoresis or Ludwig-Soret-effect and was first reported by Carl Ludwig in 1856 [1]. Over twenty years later, it was systematically investigated in electrolyte solutions by Charles Soret [2]. Soret developed phenomenological equations describing the thermodiffusion based on Fick's diffusion equations. In a binary mixture it can be described as a mass flux ⃗j along a temperature gradient ∇T with ⃗ j = −rD∇c−rc(1−c)DT∇T; (1.1) where DT is the thermal diffusion coefficient, r is the mass density and c is the concentration given as mass fraction [3]. Along the concentration gradient ∇c that arises from the ther- modiffusion, Fickian diffusion takes place characterised by the diffusion coefficient D. With a stable temperature gradient, a steady state is reached where Fickian and thermodiffusion flux cancel each other out and ⃗j becomes zero. Then the concentration gradient over the temperature gradient is proportional to a constant value D 1 Dc S ≡ T = − ; (1.2) T D c(1−c) DT −1 which is defined as the Soret coefficient ST with a unit of K . The thermophobic component of the mixture, which enriches at the cold side, has a positive Soret coefficient, while the thermophilic one enriches on the warm side and has a negative ST. 2 Chapter 1: Introduction Figure 1.1: Contributions to the Soret effect. 1.1.1 Contributions to the Soret effect Thermodiffusion has been observed in mixtures of any kind: in homogenous gases, liquids and solids as well as in heterogeneous mixtures like gas bubbles in liquids, colloids and aerosols [4,5]. Experimental findings show that the thermodiffusion behaviour of a substance is sensitive to a large number of parameters. The main factors are illustrated in Fig.