Streptavidins with intersubunit crosslinks have enhanced stability.
Natural tetrameric streptavidin has two subunit interfaces; one is a strong interface between subunits in a tightly associated dimer, and the other is a weak interface between a pair of such dimers (dimer-dimer interface). To test whether strengthening the weak dimer-dimer interface could provide streptavidin with additional structural stability, covalent crosslinks were introduced between adjacent subunits through the dimer-dimer interface. Specific crosslinking sites were designed by site-directed mutations of His-127 residues that are in close proximity in natural streptavidin. The first and second streptavidin constructs have a disulfide bond and an irreversible covalent bond, respectively, between two Cys-127 residues across the dimer-dimer interface. The third variant is a hybrid tetramer consisting of two different streptavidin species, one having lysine and the other aspartic acid at position 127, which are covalently crosslinked. All streptavidin constructs with intersubunit crosslinks showed higher biotin-binding ability than natural core streptavidin after heat treatment. All of these crosslinked streptavidins retained bound biotin more stably than natural core streptavidin in guanidine hydrochloride at very acidic pH. These results suggest that the introduction of covalent bonds across the dimer-dimer interface enhances the overall stability of streptavidin.
Reznik
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GO
Center for Advanced Biotechnology, Boston University, MA 02215, USA.
Vajda
S
S
Smith
C L
CL
Cantor
C R
CR
Sano
T
T
D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004220:Disulfides / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D004220:Disulfides; D011994:Recombinant Proteins; D019809:Streptavidin; D019791:Guanidine