10.1021/pr800923e.s059 Deborah R. Francoleon Deborah R. Francoleon Pinmanee Boontheung Pinmanee Boontheung Yanan Yang Yanan Yang UnMi Kim UnMi Kim A. Jimmy Ytterberg A. Jimmy Ytterberg Patricia A. Denny Patricia A. Denny Paul C. Denny Paul C. Denny Joseph A. Loo Joseph A. Loo Robert P. Gunsalus Robert P. Gunsalus Rachel R. Ogorzalek Loo Rachel R. Ogorzalek Loo S-layer, Surface-Accessible, and Concanavalin A Binding Proteins of <i>Methanosarcina acetivorans</i> and <i>Methanosarcina mazei</i> American Chemical Society 2009 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 00:00:00 Dataset 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_/2867137 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.