Study of structural features and aggregation of S-layer proteins from lactobacilli.

BOLLA, PATRICIA ARACELI; PERRUZO PABLO JOSÉ; CASELLA MÓNICA LAURA; SERRADELL, MARÍA DE LOS ANGELES

Campinas

Congreso; The LNLS 26th Annual Users? Meeting (RAU); 2016

CNPEM/LNLS

Crystalline S-layers are the outermost cell envelope components of many bacteria and archaea. S-layers are monomolecular arrays composed of identical protein or glycoprotein subunits with molecular weights ranging from 40 to 200 kDa. Isolated S-layer proteins (SLP) show an intrinsic tendency to reassemble into regular arrays after removal of the disrupting agent used in the extraction procedure. They reassemble spontaneously in solution and on solid supports by an entropy-driven process [2]. The monomers of the S-layer SbpA of Bacillus sphaericus, are believed to consist of three cell wall anchoring domains followed by a calcium binding domain and three C-terminal immunoglobulin-like domains [4]. The self-assembly products generated in suspension may have the form of flatsheets, open-ended cylinders or closed vesicles. Different environmental parameters such as temperature, pH, ion composition, and/or ionic strength, could lead to formation of different self-assembly products, such as flat sheets, open-ended cylinders, or closed vesicles [2,3]. In these sense, SAXS measurements on S-layer isolated from three different strains of Lactobacillus kefiri with or without calcium were performed in order to evaluate the aggregation of these proteins.The S-layer proteins were obtained from three different strains of Lactobacillus kefiri. For the extraction from bacterial cells, the microorganisms were treated with 5 M LiCl and it were centrifuged (16000 g at 10 °C for 30 min). The supernatant was filtered through a membrane of 0.45 μm diameter pore. The solution containing the S-layer protein was dialyzed against PBS for 24 h at room temperature, making 3 changes of 1L each, using a cellulose membrane (SpectraPor membrane tube, MWCO 12000,SIGMA-Aldrich). Protein concentration was determined by the Bradford method.In this work, SAXS measurements S-layers were performed using the SAXS1 beamline at the LNLS (Campinas, Brazil) using a wavelength 1.55 Å; exposure time of 200 sec.; sample detector distance of 1 m). In the first step the S layers in PBS were studied. In a second step the protein aggregation in presence of Ca+2 (10mM in PBS buffer solution) was evaluated.Fig. 1 shows the scattering curves and distance distribution functions p(r) of three different S-layer proteins of Lactobacillus kefiri. The scattering behavior of these proteins appeared to be quite different. SAXS curves varied for each sample, suggesting the presence of some differences in the protein architecture depending on the strain. In addition, the scattering patterns of each S-Layer protein were significantly different in absence or presence of Ca+2.The parameters RG, dmax, and V, obtained from SAXS curves are listed in Table 1. These parameters confirmed the differences suggested by SAXS curves for each S-Layer protein in absence or presence of Ca+2 . Moreover, the distribution function P(R) of SAXS data showed substantial variations when analyzed using the GNOM program, such as changes in dmax upon addition of Ca+2. In particular, dmax of S-Layer protein from Lb. kefiri 8348 increased (from 20,35nm to 42,27nm) and dmax of S-Layer protein from Lb. kefiri 5818 decreased (from 36,45nm to 11,07nm), meanwhile the S-Layer protein from Lb. kefiri 83111 did not show significant changes when Ca+2 was added.SAXS data were used to generate DAMMIN models of three-dimensional arrangements of scattering centers. The S-Layer protein of Lb. kefiri 8348 in PBS buffer self-assembled as a compact hexamer. In the presence of Ca+2 the structure showed a core and slender arms radiating outward and extending towards one face of the core (Fig. 2). The hexamer structure obtained for this protein is in agreement with the protein arrangements observed by AFM for this sample [Gerbino E.; Doctoral Thesis, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 2011]. Three different S-Layer proteins isolated from three different Lb. kefiri strains were studied for the first time by SAXS. Results obtained allowed to evaluate the unknown structure of these proteins, and different behaviors were observed in the presence of Ca+2 (increase, decrease or unchange in size). Research is in progress to evaluate these changes.