S-layer proteins from lactobacilli: characterization by FTIR and DSC

MOBILI P, GÓMEZ-ZAVAGLIA A, LONDERO A, ROSEIRO T, EUSEBIO E, DE ANTONI G AND RUI FAUSTO

Buenos Aires

Workshop; International Workshop on Infrared Spectroscopy Applied to Biological and Biomimetic Systems: From the Isolated Molecule to the Cell; 2007

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 and the changes induced on these structures after heating. 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. The infrared spectra, in the 4000-500 cm-1 range, were recorded by transmission spectroscopy of solid proteins in KBr pellets, using a variable temperature infrared cell connected to a digital controller. Thermal studies were carried out in a Perkin Elmer DSC7 calorimeter, over the 15-130ºC range, with scanning rate 25ºC/min. Second derivative spectral analysis and peak-fitting showed 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-layer proteins showed b-sheet contents ranging from 20 to 40% and a-helix contents from 10 to 20%. DSC analysis showed transitions at 58 and 98ºC for L. brevis S-layer and at 69 and 115ºC for L. kefir S-layers. Transitions observed by DSC were in agreement with the ones observed by the second derivative analysis of FTIR spectra obtained at different temperatures.