STRUCTURAL ANALYSIS OF THE INTERACTION S-LAYER-METALS BY MEANS OF FTIR SPECTROSCOPY

GERBINO, E.; MOBILI, P; TYMCZYSZYN E.; FAUSTO RUI; GOMEZ ZAVAGLIA, A.

Manchester

Congreso; SPEC2010; 2010

SPEC

S-layers are macromolecular paracrystalline arrays commonly found in bacteria. These porous structures completely cover the bacterial cell, representing the outermost interaction surface with their environment. Several specific functions have been assigned to S-layers from different microorganisms, e.g.,  protective coats, molecular sieves, molecule and ion traps, cell adhesion and surface recognition, virulence factors. The S-layer is composed of protein subunits assembled in lattices with different organization, attached to the underlying cell wall by non-covalent bonds. They can be disassembled and solubilized into protein monomers by dissociating agents, such as 5 M LiCl. S-Layer protein subunits show the intrinsic tendency to reassemble into two-dimensional arrays after the removal of the disrupting agent used in the extraction. The tendency of S-layer proteins to reassemble spontaneously in an ordered pattern, together with their ability to bind different molecules or ions, has converted S-layers in an attractive target for biophysical studies and structural research, particularly regarding their potential application in nanotechnology and bioremediation [1-3]. However, information regarding the molecular basis of the (S-layer)-(metal ion) interactions and their effects on the structure of S-layer proteins is still very scarce. For this reason, in the present work we studied the interaction between lactobacilli S-layer proteins and heavy metal ions and their structural implications. Two strains of Lactobacillus kefir (JCM 5818 and CIDCA 8348), known to bear structurally different S-layer proteins [4,5] were used. S-layer proteins were extracted using 5 M LiCl, extracts were concentred by ultrafiltration, dialyzed against bidistilled water and lyophilized. Then, 1 mg of each lyophilized S-layer protein was resuspended in 1 mL of ultrapure water (milli-Q) and incubated with 0.3 mM of Pb(NO3)2, Cd(NO3)2, Ni(NO3)2.6H2O, Zn(NO3)2.6H2O or Al(NO3)3.9H2O at 30 ºC for 24 hours. The suspensions were centrifuged at 6600 x g for 5 min. and pellets were preserved. Aliquots of pellets were transferred onto a CaF2 optical plate, and dried at 45 ºC for 15 min. The resulting transparent films were directly used for FT-IR spectroscopy. S-layer proteins treated in the same way in absence of metals were used as control.  A total of 128 IR scans were coadded, at 4 cm-1 resolution. Inverted second derivative spectra were used to estimate the number, position and relative contribution of individual elements composing Amide I band (1600-1720 cm-1), and this information was taken into account to fit Amide I bands in protein spectra with Gaussian band profiles. This approach allowed for the assignment of the S-layer secondary structures. The incubation of S-layer proteins from L. kefir JCM 5818 and L. kefir CIDCA 8348 with different metal ions produced changes in their secondary structure, as a result of the interaction between proteins and ions. Such changes might be potentially useful for use of this type of systems in bioremediation.   References [1]  U.B. Sleytr, P. Messner, P. Pum and M. Sára. Angew. Chem. Int. Ed. 38, 1034-1054 (1999). [2]  U.B. Sleytr, M. Sara, D. Pum and B. Schuster. Prog. Surf. Sci. 68, 231-278 (2001). [3]  V.G. Debabov. Mol. Biol. 38, 482-493 (2004). [4]  P. Mobili, A. Londero, T. Roseiro, E. Eusebio, G. De Antoni, R. Fausto and A. Gómez-Zavaglia, Vibrat. Spectrosc. 50, 68-77 (2009). [5]   P. Mobili, M. Serradell, S. Trejo, F. Avilés Puigvert, G. Abraham and G. De Antoni, Ant. van Leeuw. 95, 363-372 (2009).