Paving the Way for Biocatalytically Active Protein Membranes
Biocatalytically active proteins (enzymes) are increasingly used in many different industrial branches and might even have bigger potential in future processes1. Therefore, further improvement of biocatalytical activities might result in an even higher efficiency of industrial processes. One possibility for this is the reduction of diffusion time in enzyme cascades. The goal of our iGEM project was to achieve increased biocatalytic efficiency by reducing the diffusion times in enzyme cascades while also give a scaffold for enzyme fixation. By using S-Layer proteins, we worked on constructing a “biocatalytically active membrane”.
What are S-Layer proteins…?
Surface-layers (S-layer) are prokaryotic protein membranes which are capable of self-assembling into two- dimensional lattices. S-layer proteins consist of two different domains2: The SLH-motive domain3, which attaches to the cell wall polymers via hydrophobic interactions and the self-assembly domain (Fig. 1), 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) (Fig3a).
Figure 1. Schematic picture of S-Layer Proteins.
S-Layer proteins attached to (A) gram-positive and (B) gram-negative bacteria.
…and why do we want to use them?
The principle of self-assembly does also transfer to in vitro experiments where S-layer proteins assemble either to different structures in solution but can also bind to surfaces (Fig 2.). We see great potential in the application of S-layers in industrial processes by covering two major improvements to biocatalysis: reduction of diffusion lengths and immobilization of enzymes. More specifically two proteins might be brought in close proximity after self-assembly while also being fixating on newly assembled surface, if attached to S-layer proteins through covalent or non-covalent binding. However, only few advances in this direction have been made in the past. Therefore, we decided to work on the foundational advance of S-Layers while also take to account existing knowledge such as S-Layer Streptavidin fusions4.
Figure 2. Re-assembly of S-Layer proteins.
Assembly of S-Layer proteins occur on cell walls, in solution, surfaces or on hydrophilic/hydrophobic membranes.
Mixture of different S-Layer types for the creation of new patterns
In our studies we want to investigate the behavior of different types of S-layer in mixture (Fig 3b). Therefore, different mixtures of the proteins SbsB (p1) from Geobacillus stearothermophilus5, PS2 (p2) from Corynebacterium glutamicum6 and RsaA (p3) from Caulobacter crescentus7 are tested under different conditions. Structural analysis of the novel surface patterns on surfaces and in solution are investigated through electron microscopy, atomic force microscopy and light scattering. For a preliminary prediction of S-layer assembly we use Monte-Carlo-Markov-chains simulation.
Figure 3. Slayer symmetries.
(A) S-Layer proteins form patterns with different symmetries. (B) Presumable new structures as result f S-Layer mixture of p1 and p3 patterns.
S-Layer fusion proteins
To explore novel potential applications, S-layer proteins were conjugated with Streptavidin4. Thus, various biotinylated fluorescence markers can be applied for FRET analysis. This can serve as model system for our premise that different attachments on the S-Layer are in close proximity to one each other. Other ideas of chimeric S-Layer proteins involve PS2 of C. glutamicum. Due to the simplicity of PS2 isolation, functional fusion proteins on the outside of the cell will be created. Here, a FRET analysis will be used as well to proof our concept, however, through a covalent fusion with CFP and YFP in the middle of the protein.
Creation of three-dimensional lattices using S-Layer Streptavidin fusion proteins.
The aforementioned S-Layer-Streptavidin proteins will be used to create three-dimensional lattices by stacking multiple S-Layer-Streptavidin with biotin linker over a process similar to atomic layer deposition (ALD) (Fig. 4). Through the pore formed by assembling S-Layer proteins is also expanded in the third dimension. The length of this pore can be determined precisely and can be used for the creation of inorganic nanotubes by using the functional side-chains of the protein as template.
Figure 4. 3D S-Layer.
S-Layer proteins (green) conjugated with Streptavidin monomers (orange) Biotin linkers (blue) attached to streptavidin causes layering of S-Layer into the third dimension.
1. Singh, R., Kumar, M., Mittal, A. & Mehta, P. K. Microbial enzymes: industrial progress in 21st century.
6, 1–15 (2016).
2. Thomas, S., Austin, J. W., McCubbin, W. D., Kay, C. M. & Trust, T. J. Roles of structural domains in the morphology and surface anchoring of the tetragonal paracrystalline array of Aeromonas hydrophila. Biochemical characterization of the major structural domain. J. Mol. Biol. 228, 652–661 (1992).
3. Ries, W., Hotzy, C., Schocher, I., Sleytr, U. B. & Sára, M. Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer. J. Bacteriol. 179, 3892–8 (1997).
4. Moll, D. et al. S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays. Proc. Natl. Acad. Sci. 99, 14646–14651 (2002).
5. Totten, R. K. et al. Catalytic solvolytic and hydrolytic degradation of toxic methyl paraoxon with la(catecholate)-functionalized porous organic polymers. ACS Catal. 3, 1454–1459 (2013).
6. Peyret, J. L. et al. Characterization of the cspB gene encoding PS2, an ordered surface‐layer protein in Corynebacterium glutamicum. Mol. Microbiol. 9, 97–109 (1993).
7. Amat, F. et al. Analysis of the intact surface layer of Caulobacter crescentus by cryo-electron tomography. J. Bacteriol. 192, 5855–5865 (2010).