Characterization of S-layer proteins from Lactobacillus by FTIR spectroscopy

P. MOBILI, A. GOMEZ-ZAVAGLIA, A. ABRAHAM, G. DE ANTONI, R. FAUSTO.

París, Francia

Congreso; XIIth European Conference on the Spectroscopy of Biological Molecules.; 2007

Université de Paris XIII

Bacteria of genus Lactobacillus are commonly found in dairy products, and are usually associated with the beneficial effects of these products to human health. This beneficial activity is at least partially related with bacterial surface properties as adhesion and aggregation. Some lactobacilli bear an S-layer, a crystalline bidimensional array of glycoproteic subunits linked non-covalently to each other and to the underlying cell-wall. As the outermost envelope in lactobacilli, S-layer could play an important role in the surface properties of these bacteria. Structural differences in S-layer proteins could be responsible for the observed variability in surface properties of different strains of lactobacilli.  FTIR has proved to be a useful tool for the study of secondary structure of proteins by means of peak-fitting and/or analysis of second derivative spectra of amide I region (1720 – 1600 cm-1). Empirical association was made between position of amide I band and/or position and relative areas of component bands in peak-fitting and the type and relative content of secondary structure in the protein. In the present work we used FTIR to study the secondary structure of S-layer proteins from different strains of lactobacilli. We studied S-layer proteins from reference strains L. brevis ATCC 8287 and L. kefir JCM 5818, and from four strains of L. kefir isolated from kefir at CIDCA (L. kefir CIDCA 83113, CIDCA 8321, CIDCA 8344 y CIDCA 8348). These bacterial strains showed differences in surface properties and in S-layer protein molecular weight, glycosilation and pattern of recognition by antibodies. S-layer proteins were extracted from bacteria using LiCl 5M, extracts were concentred by ultrafiltration, dialyzed against bidistilled water and lyophilized. FTIR analysis was made by transmission spectroscopy of solid proteins in KBr pellets (ratio 1mg protein / 100mg KBr). A total of 512 scans were coadded at a 4cm-1 resolution. In unresolved spectra, amide I band of L. brevis  S-layer is centred at 1645cm-1, showing a high b-sheet content, while L. kefir S-layers presented amide I bands centred in the range from 1650 to 1660cm-1, suggesting a higher content of a-helix. Second derivative spectral analysis and peak-fitting show that L. brevis S-layer has a b-sheet content of approximately 50% and no a-helix, in good agreement with predictions based on its primary structure. The same analysis on L. kefir S-layers showed b-sheet contents of around 50% and higher a-helix contents, ranging from 11 to 20 %.