Supramolecular design of regularly structured S-layer protein lattices with chemical specificity and orientation

Research project P 14689 Supramolecular design of functionalized S-layer lattices Margit SRA 09.10.2000 Crystalline bacterial cell surface layer (S-layers) represent the outermost cell envelope component in many bacteria and archaea and they are composed of a single protein or glycoprotein species with the ability to self-assemble into 2-D crystalline arrays. Isolated S-layer subunits frequently recrystallize into closed monolayers on solid supports (noble metals, silicon wafers, glass), on spread lipid films and on liposomes. Due to the anisotropic, surface characteristics of the outer and inner surface of the S-layer subunits, the orientation of the recrystallized S-layer monolayer strongly depends on the properties of the supports and interfaces (e.g. hydrophobicity and charge) and on the experimental conditions (e.g. pH value, ionic strength and ionic composition). This is a severe drawback if recombinant functional S-layer fusion proteins or recombinant S-layer proteins with inserted functional peptide sequences are used for recrystallization for generating regularly structured affinity monolayers. Since in bacterial cells the N-terminal part of the S-layer subunits, mostly possessing three typical S-layer homologous (SLH) motifs, recognizes as the proper anchoring structure in the rigid cell wall layer a distinct type of secondary cell wall polymer, the latter shall be exploited for functionalization of various types of supports to achieve an oriented binding of recombinant functional S-layer proteins. This implies that secondary cell wall polymers or their biologically active degradation products which may represent the smallest functional unit are either linked to solid supports, to lipid head groups in liposomes or that glycolipids synthesized of secondary cell wall polymers or of appropriate degradation products and lipid molecules are incorporated into lipid monolayers or into liposomes. By applying the surface plasmon resonance technique, the affinity constants between the N-tenninal part as well as between the individual SLH-motifs and the corresponding type of secondary cell wall polymer will be determined. Derived from these results, recombinant low or high affinity S-layer proteins will be.constructed by novel combinations of selected SLH-motifs - a strategy that should, allow to. modulate the stability of generated 3-D supramolecular structures. To investigate whether the C-terminal amino acids of the full-length S-layer proteins and of C-terminal self-assembling truncations are. located on the outer surface of the S-layer lattice, the sequence encoding the strept-tag will be linked to the sequences encoding the various S-layer protein forms. The surface, accessibility of the step-tag will be checked by applying streptavidin-conjugates to oriented S-layer monolayers, formed by the different recombinant S-layer proteins on supports functionalized with secondary cell wall polymer. An alternative strategy for identifying surface located amino acid postions can be seen in the insertion of cysteine and strep-tag 11 into t h a t segment of the C-terminal part of the fulllength S-layer proteins which is not required for the self-assembly process and for generation of the regular lattice structure. This strategy should enable to screen tw~o independent amino acid positions within a single type of mutant protein. Surface-located amino acid positions shall finally be exploited for (multiple) insertion(s) of functional peptide sequences and / or for construction of S-layer fusion proteins as required for generating biochips, affinity (separation) and targeting structures, as well as for biomimetic immunogens. Furthermore, 3-D crystallization of overlapping water soluble N- and C-terminal truncations will be performed for complete structural (X-ray) analysis of S-layer proteins. This shall finally allow to correlate the structure of an inserted peptide sequence as presented in a heterologous, environment with its functional properties.

Crystalline bacterial cell surface layer (S-layers) represent the outermost cell envelope component in many bacteria and archaea and they are composed of a single protein or glycoprotein species with the ability to self-assemble into 2-D crystalline arrays. Isolated S-layer subunits frequently recrystallize into closed monolayers on solid supports (noble metals, silicon wafers, glass), on spread lipid films and on liposomes. Due to the anisotropic surface characteristics of the outer and inner surface of the S-layer subunits, the orientation of the recrystallized S-layer monolayer strongly depends on the properties of the supports and interfaces (e.g. hydrophobicity and charge) and on the experimental conditions (e.g. pH value, ionic strength and ionic composition). This is a severe drawback if recombinant functional S-layer fusion proteins or recombinant S-layer proteins with inserted functional peptide sequences are used for recrystallization for generating regularly structured affinity monolayers. Since in bacterial cells the N-terminal part of the S-layer subunits, mostly possessing three typical S-layer homologous (SLH) motifs, recognizes as the proper anchoring structure in the rigid cell wall layer a distinct type of secondary cell wall polymer, the latter shall be exploited for functionalization of various types of supports to achieve an oriented binding of recombinant functional S-layer proteins. This implies that secondary cell wall polymers or their biologically active degradation products which may represent the smallest functional unit are either linked to solid supports, to lipid head groups in liposomes or that glycolipids synthesized of secondary cell wall polymers or of appropriate degradation products and lipid molecules are incorporated into lipid monolayers or into liposomes. By applying the surface plasmon resonance technique, the affinity constants between the N-terminal part as well as between the individual SLH-motifs and the corresponding type of secondary cell wall polymer will be determined. Derived from these results, recombinant low or high affinity S-layer proteins will be constructed by novel combinations of selected SLH-motifs - a strategy that should allow to modulate the stability of generated 3-D supramolecular structures. To investigate whether the C-terminal amino acids of the full-length S-layer proteins and of C-terminal self-assembling truncations are located on the outer surface of the S-layer lattice, the sequence encoding the strept-tag will be linked to the sequences encoding the various S-layer protein forms. The surface accessibility of the step-tag will be checked by applying streptavidin-conjugates to oriented S-layer monolayers formed by the different recombinant S-layer proteins on supports functionalized with secondary cell wall polymer. An alternative strategy for identifying surface located amino acid postions can be seen in the insertion of cysteine and strep-tag II into t h a t segment of the C-terminal part of the full-length S-layer proteins which is not required for the self-assembly process and for generation of the regular lattice structure. This strategy should enable to screen two independent amino acid positions within a single type of mutant protein. Surface-located amino acid positions shall finally be exploited for (multiple) insertion(s) of functional peptide sequences and / or for construction of S-layer fusion proteins as required for generating biochips, affinity (separation) and targeting structures, as well as for biomimetic immunogens. Furthermore, 3-D crystallization of overlapping water soluble N- and C-terminal truncations will be performed for complete structural (X-ray) analysis of S-layer proteins. This shall finally allow to correlate the structure of an inserted peptide sequence as presented in a heterologous environment with its functional properties.