S-layer, Surface-Accessible, and Concanavalin A Binding Proteins of Methanosarcina acetivorans and Methanosarcina mazei
datasetposted on 03.04.2009, 00:00 authored by Deborah R. Francoleon, Pinmanee Boontheung, Yanan Yang, UnMi Kim, A. Jimmy Ytterberg, Patricia A. Denny, Paul C. Denny, Joseph A. Loo, Robert P. Gunsalus, Rachel R. Ogorzalek Loo
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 Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo 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 N2 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 M. acetivorans C2A and M. mazei 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.
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study surface proteinsMA 0829N 2amines reactivepH mediasurface layerBinding ProteinsMethanosarcina acetivoransStreptavidin affinity enrichmentMMMethanosarcina mazei100 proteinsacylation chemistryglycosylated formsarchaeal species Methanosarcina acetivorans C 2AMethanosarcina mazeiThe outermost cell envelope structureproteinaceous latticewash conditionsalternative strategyvivo biotinylation methodologyacetivorans C 2A