The Atomic Resolution Structure of S-Layer Proteins

Crystalline bacterial surface layers (S-layers) are one of the most common surface structures in prokaryotic cells. In spite of their biological importance for the functionality of prokaryotic cells and the potential applications in nanobiotechnology, high resolution structural information of S-layer proteins is scarce. The main reason for the absence of 3-D structural information resides in the tendency of S-layer proteins to self-assemble into 2-D crystalline lattices, thereby preventing the formation of 3-D crystals. In the framework of this project the molecular structure of selected S-layer proteins shall be elucidated for understanding their biological function and as a basis for applications in nanobiotechnology. In order to obtain 3-D crystals suitable for crystallographic structure determination, three strategies will be pursued. In the first strategy, N- or C-terminal truncations of the S-layer proteins SbsC, SbsB and SbpA will be used, which have been shown to be inactive in self-assembly. By applying this approach, crystals of one N-terminal and one C-terminal truncation mutant of the S-layer protein SbsC from Geobacillus stearothermophilus ATCC 12980 have been obtained already. The C-terminal truncation mutant rSbsC(31-844) yielded crystals diffracting to 3 Å resolution and native and derivative data sets have been collected (Pavkov, Oberer et al. 2003). In the second approach, cysteine mutants will be employed, whereas in the third approach conditions under which the full-length S-layer proteins are inactive in self-assembly will be used to obtain 3-D crystals. Such conditions have been identified for the S-layer protein SbpA from Bacillus sphaericus CCM 2177, which is devoid of self-assembly in the absence of calcium ions and in the presence of the secondary cell wall polymer (SCWP). Crystallizing the S-layer protein together with a ligand mimicking the SCWP and determining the complex structure will be an important step towards elucidating the functional role of this interaction.

Crystalline bacterial surface layers (S-layers) are one of the most common surface structures in prokaryotic cells. In spite of their biological importance for the functionality of prokaryotic cells and the potential applications in nanobiotechnology, high resolution structural information of S-layer proteins is scarce. The main reason for the absence of 3-D structural information resides in the tendency of S-layer proteins to self-assemble into 2-D crystalline lattices, thereby preventing the formation of 3-D crystals. In the framework of this project the molecular structure of selected S-layer proteins shall be elucidated for understanding their biological function and as a basis for applications in nanobiotechnology. In order to obtain 3-D crystals suitable for crystallographic structure determination, three strategies will be pursued. In the first strategy, N- or C-terminal truncations of the S-layer proteins SbsC, SbsB and SbpA will be used, which have been shown to be inactive in self-assembly. By applying this approach, crystals of one N-terminal and one C-terminal truncation mutant of the S-layer protein SbsC from Geobacillus stearothermophilus ATCC 12980 have been obtained already. The C-terminal truncation mutant rSbsC(31-844) yielded crystals diffracting to 3 Å resolution and native and derivative data sets have been collected (Pavkov, Oberer et al. 2003). In the second approach, cysteine mutants will be employed, whereas in the third approach conditions under which the full-length S-layer proteins are inactive in self-assembly will be used to obtain 3-D crystals. Such conditions have been identified for the S-layer protein SbpA from Bacillus sphaericus CCM 2177, which is devoid of self-assembly in the absence of calcium ions and in the presence of the secondary cell wall polymer (SCWP). Crystallizing the S-layer protein together with a ligand mimicking the SCWP and determining the complex structure will be an important step towards elucidating the functional role of this interaction.