Francoleon, Deborah R. Boontheung, Pinmanee Yang, Yanan Kim, UnMi Ytterberg, A. Jimmy Denny, Patricia A. Denny, Paul C. Loo, Joseph A. Gunsalus, Robert P. R. Ogorzalek Loo, Rachel S-layer, Surface-Accessible, and Concanavalin A Binding Proteins of <i>Methanosarcina acetivorans</i> and <i>Methanosarcina mazei</i> The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often post-translationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over 100 proteins were annotated to be S-layer components in the archaeal species <i>Methanosarcina acetivorans</i> C2A and <i>Methanosarcina mazei</i> Gö1, reflecting limitations of current predictions. An <i>in vivo</i> biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N<sub>2</sub> fixing conditions, thus, minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed nonspecifically bound proteins. This methodology revealed S-layer proteins in <i>M. acetivorans</i> C2A and <i>M. mazei</i> Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically localized. This approach provides an alternative strategy to study surface proteins in the archaea. study surface proteins;MA 0829;N 2;amines reactive;pH media;surface layer;Binding Proteins;Methanosarcina acetivorans;Streptavidin affinity enrichment;MM;Methanosarcina mazei;100 proteins;acylation chemistry;glycosylated forms;archaeal species Methanosarcina acetivorans C 2A;Methanosarcina mazeiThe outermost cell envelope structure;proteinaceous lattice;wash conditions;alternative strategy;vivo biotinylation methodology;acetivorans C 2A 2009-04-03
    https://acs.figshare.com/articles/dataset/S_layer_Surface_Accessible_and_Concanavalin_A_Binding_Proteins_of_i_Methanosarcina_acetivorans_i_and_i_Methanosarcina_mazei_i_/2867122
10.1021/pr800923e.s050