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.