Diffusion of proteins inside crowded structures generated using microemulsions

Microemulsions are thermodynamically stable mixtures of water, oil and surfactant. In the case of microemulsions based on sugar surfactants a cosurfactant as an additional component is necessary, most often a short-chain alcohol. In this thesis mainly pure surfactants are used. The focus is on n-nonyl-ß-d-maltosid and n-dodecyl-ß-d-maltosid as model systems for microemulsions based on technical grade surfactants. Depending on the ratio of the components microemulsion form different structures. In the bicontinuous phase continuous oil and water domains are present separated by a surfactant/co-surfactant film. At higher surfactant amounts the bicontinuous structure passes over to a droplet phase. The size of these structures is in the scale of up to 100nm, therefore microemulsions look transparent for the human eye. Bicontinuous microemulsions are a promising carrier medium for decontamination applications. Chemical warfare agents are mainly lipophilic, in contrast degradation agents are hydrophilic. A microemulsion is able to solubilize lipophilic and hydrophilic molecules. At the interface the warfare agent is close to the degradation agent and can be eliminated. The protein diisopropyl-fluorophosphatase (DFPase) is a promising candidate for decontamination applications and is able to degrade different warfare agents. Therefore, the knowledge of the dynamics and properties of enzymes inside a bicontinuous structure is of big importance. In this thesis different microemulsion systems based on C9G2 or C12G2, water, cyclohexane and 1-pentanol are systematically characterized by X-ray and neutron scattering experiments. By using an improved Green Fluorescent Protein (GFP+) as a counterpart of DFPase with a similar size, the diffusion inside a microemulsion can be studied with the fluorescence correlation spectroscopy (FCS) method. Here, fluorescent impurities in the used components are a problem. By the choice of a suitable concentration of GFP+ and regarding the dynamics of the microemulsion structure, it could be shown that the protein is able to move inside the microemulsion structure. Hence, microemulsions are interesting model systems to produce crowding effects in a controlled way. This is probably the most important result of this thesis. In the C9G2 system a bicontinuous phase is present. At small oil/water ratios o/w with a high water amount big water domains exist with length scales of approximately 10nm, which allows protein diffusion. With increasing oil/water ratio the protein diffusion is more and more hindered. This results in an increase of the diffusion time and a decrease of the anomalous diffusion exponent. At even higher oil/water ratios the diffusion of the protein is nearly identical with the microemulsion dynamics. The length scales are too small to allow protein diffusion, therefore it is stuck in the structure. The C12G2 system shows an oil-in-water droplet phase which also hinders the GFP+ diffusion due to crowding effects. For small oil/water ratios nearly free diffusion is present, at o/w = 0.3 - 0.5 the diffusion is hindered, the anomalous diffusion exponent decreases. At o/w = 0.6 the protein diffusion reflects the microemulsion dynamics. Again at higher values of o/w, GFP+ is stuck in the structure because of the high oil fraction. Two microemulsion systems with different structures were studied, which allow protein diffusion due to their length scales. These results are related to applications: For the formulation of decontamination media the knowledge of the dynamics of the decontaminating protein is of importance. Moreover, pure surfactants can be easily replaced by technical grade surfactants. This enables cheap systems for a production-scale. Furthermore, the situation in living cells can be simulated by a microemulsion structure, where a confined diffusion is present as well. Additional neutron spin echo experiments emphasize the complexity when studying a microemulsion system based on four components. The contrast matching procedure, which is necessary when protein dynamics inside the microemulsion are investigated, is very difficult and requires a high amount of surfactant.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bayreuth

Year

2013

Author dc:creator

• Neubauer, Ralph

Contributors dc:contributor

• Förster, Stephan

Repository record source_url

https://epub.uni-bayreuth.de/id/eprint/88/

OAI identifier oai:identifier

oai:epub.uni-bayreuth.de:88