Improvement of Biocatalytic Properties
by structured S-Layer-Proteins
The purpose of this project is the improvement of biocatalytic properties of multi-step reactions by fixation of enzymes to surface-layer (S-layer) proteins.
The p2 S-layer protein PS2 from Corynebacterium glutamicum with a p2-symmetry implies proximity itself. After proofing this by performing a FRET assay we use this proximity for reduction of diffusion lengths in multi-step reactions . The p2 S-layer should induce proximity of two proteins coupled with S-layer proteins (fusion-proteins) after reassembly. For a proof of concept, a model reaction for enzymatic coupling via S-layer proteins will be created by fusing two enzymes to it. The first one is invertase which is capable of cleaving sucrose into glucose and fructose. The second enzyme is the glucose phosphate kinase hexokinase. This reaction will result in the production of glucose-6-phosphate which can be measured by a simple optical test. We expect a significant increase of the reaction rate due to the reduced diffusion length. This proof of concept will be reinforced by computer simulations with a molecular dynamic simulation of the enzymatic reactions on the S-layers.
Mixing S-layer proteins with different properties of symmetry (p1, p2 and p3) form novel surface patterns with different lattice symmetries. Of specific interest is whether the proteins form domains or a mixture of the different structures. The results will be supported by Monte-Carlo simulation of the S-layer mixture.
Furthermore, we plan to create a three-dimensional structure using S-layer streptavidin fusion proteins which will be attached to a hydrophobic membrane due to amphipathic properties of the S-layer proteins. The so formed lattices can be linked to form a three-dimensional super-lattice using synthetic biotin-linkers. These steps can be repeated multiple times causing the super-lattice to grow and form a nano-tube, for example.
What are S-Layer proteins?
Surface-layers (S-layer) are prokaryotic protein membranes which assemble as two-dimensional symmetric lattices (see figure).
S-layer proteins have two different domains: The SLH-motive domain, which attaches non-covalent to the cell wall polymers, and the self-assembly domain, which interacts hydrophobic as well as hydrophilic with other S-layer proteins. For this project the structural properties are of specific interest: S-layer proteins form two-dimensional lattices with different symmetries: oblique (p1,p2), square (p4), hexagonal (p3,p6) (see figure).
Why did we choose this particular project?
S-layer proteins have a great application potential in biotechnology, biomedicine and synthetic biology due to their capability of self-assembly. The fixation of enzymes and the improvement of biocatalytic properties of multi-step reactions has auspicious advantages for biosensors and biocatalysts. Furthermore, nano-tubes are sought-after in many different fields of research. Not negligible is the fact, that S-layers are easy to isolate and therefore a economical scope of application and effective field of research.
What do we aim to achieve?
Our overarching goal is the improvement of biocatalytic properties in multi-step reactions. By fixation of enzymes on S-layer proteins we want to reduce the diffusion lengths of substrates between enzymes in multi-step reactions. By this arrangement of different enzymes in close proximity, we predict an improvement of the bio-catalytic activity in multi-step reactions. For a proof-of-concept we are using Invertase and Hexokinase on the p2 S-layer PS2 from Corynebacterium glutamicum. Furthermore, we work on the Stacking of identical monolayers, which can be used as a template for synthesis of inorganic nanotubes.
Mixture of S-layer proteins in 2D-structure
We plan structural analysis of the novel surface patterns on surfaces and in solution through electron microscopy, atomic force microscopy and light scattering. For a preliminary prediction of S-layer assembly we use Monte-Carlo-Markov-chains simulation.
S-layer proteins in 3D-structure
We are working on the creation of three-dimensional lattices built of S-layer fusion proteins with monomeric streptavidin. The biotin-linker can bind to Streptavidin and the following deposition of additional S-layers onto biotin molecules induces growing in the third dimension. The stacking of identical monolayers allows an exact determination of the pore length for synthesis of inorganic nanotubes. Therefore, our results can be used as a template for synthesis of inorganic nanotubes.
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