S-layer, Surface-Accessible, and Concanavalin A Binding Proteins of <i>Methanosarcina acetivorans</i> and <i>Methanosarcina mazei</i>
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
10.1021/pr800923e.s012
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_/2867029
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.
2009-04-03 00:00:00
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