Relevance of charged groups for the integrity of the S-layer from Bacillus coagulans E38-66 and for molecular interactions.
In this paper, the importance of charged amino and carboxyl groups for the integrity of the cell surface layer (S-layer) lattice from Bacillus coagulans E38-66 and for the self-assembly of the isolated subunits was investigated. Amidination of the free amino groups which preserved their positive net charge had no influence on both. On the other hand, acetylation and succinylation, which converted the amino groups into either neutral or negatively charged groups, and amidation of carboxyl groups were accompanied by the disintegration or at least by the loss of the regular structure of the S-layer lattice. Treatment of S-layer monolayers with the zero-length cross-linker carbodiimide led to the introduction of peptide bonds between activated carboxyl groups and amino groups from adjacent subunits. This clearly indicated that in the native S-layer lattice the charged groups are located closely enough for direct electrostatic interactions. Under disrupting conditions in which the S-layer polypeptide chains were unfolded, 58% of the Asx and Glx residues could be amidated, indicating that they occur in the free carboxylic acid form. As derived from chemical modification of monolayer self-assembly products, about 90% of the lysine and 70% of the aspartic and glutamic acid residues are aligned on the surface of the S-layer protein domains. This corresponded to 45 amino groups and to 63 carboxyl groups per S-layer subunit. Labelling experiments with macromolecules with different sizes and charges and adsorption studies with ion-exchange particles revealed a surplus of free carboxyl groups on the inner and on the outer faces of the S-layer lattice. Since the carboxyl groups on the outer S-layer face were accessible only for protein molecules significantly smaller then the S-layer protomers or for positively charged, thin polymer chains extending from the surface of ion-exchange beads, the negatively charged sites must be located within indentations of the corrugated S-layer protein network. This was in contrast to the carboxyl groups on the inner S-layer face, which were found to be exposed on elevations of the S-layer protein domains (D. Pum, M. Sára, and U.B. Sleytr, J. Bacteriol. 171:5296-5303, 1989).
Sára
M
M
Zentrum für Ultrastrukturforschung, Universität für Bodenkultur, Vienna, Austria.
Sleytr
U B
UB
D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000737:chemistry / Q000648:ultrastructure*; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000648:ultrastructure; D002473:Cell Wall / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000032:analysis*; D008854:Microscopy, Electron
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C055764:S-layer proteins