A streptavidin variant with slower biotin dissociation and increased mechanostability

Abstract

Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, DNA amplification and nanotechnology. We identified a mutant streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. FtsK, a motor protein important in chromosome segregation, rapidly displaced streptavidin from biotinylated DNA, whereas traptavidin resisted displacement, indicating the force generated by Ftsk translocation.

Figure 1. Traptavidin exhibits a slower off-rate from biotin-conjugates

(a) PyMOL images of residues mutated to produce traptavidin (S52G R53D) in structures of streptavidin with biotin (1mk5) and without biotin (1swa). Biotin is shown as spheres of van der Waals radius. S52 and R53 are colored green. Without biotin the L3/4 loop is disordered and does not give clear electron density. (b) Traptavidin exhibits a slower off-rate to biotin. Streptavidin and traptavidin were bound to ³H-biotin; excess non-radioactive biotin was then added and the fraction of ³H-biotin still bound was determined after varying times at 37 °C and pH 7.4. Mean of triplicate measurements ± 1 s.d. (Some error bars are too small to be visible.) (c) Traptavidin exhibits a slower off-rate to a biotin-conjugate at neutral pH. Avidin, streptavidin and traptavidin bind to biotin-4-fluorescein, quenching its fluorescence. Upon addition of excess free biotin, biotin-4-fluorescein dissociates and the increase in fluorescence is observed at 37 °C and pH 7.4. Mean of triplicate measurements ± 1 s.d. (d) Traptavidin exhibits a slower off-rate at weakly acidic pH. Streptavidin and traptavidin were analyzed as in (c) at 37 °C and pH 5.0. Mean of triplicate measurements ± 1 s.d.

Figure 2. Traptavidin shows increased thermostability

(a) Thermostability of tetramer structure. Streptavidin (upper panel) or traptavidin (middle panel) were incubated for 3 min at the indicated temperatures before SDS-PAGE and Coomassie staining. The positive control (C) was mixed with SDS before heating at 95 °C. Tetramer and monomer bands are indicated. The percentage monomer from duplicate gels is plotted in the lower panel. (b) Thermostability of biotin-conjugate binding. Streptavidin or traptavidin were incubated with biotinylated DNA and heated for 3 min at the indicated temperature before agarose gel electrophoresis and fluorescent imaging of DNA. The left lane is a negative control with no streptavidin or traptavidin added. Bands corresponding to DNA that is free or bound to streptavidin or traptavidin are marked. The percentage of biotinylated DNA free from streptavidin or traptavidin is labeled under each lane.

Figure 3. Traptavidin shows increased mechanical stability

(a) Traptavidin resists AFM displacement. Single-molecule rupture forces between a biotinylated bead and an AFM tip coated with streptavidin (black circles) or traptavidin (red squares), acquired at a range of loading rates. Means are shown ± 1 s.e.m. (streptavidin n = 400, traptavidin n = 562), with a line of best fit (solid) and 95% confidence limits to this line (dashed) in black for streptavidin and in red for traptavidin. (b) Cartoon of motor assay. DNA, containing a loading site for FtsK (αβ domains in blue, γ in gray) and a biotinylated thymidine near the terminus, was capped with traptavidin or streptavidin (green). In the presence of ATP, FtsK translocates along the DNA and collides with streptavidin or traptavidin. (c) Traptavidin resists molecular motor displacement. Displacement of streptavidin (SA) or traptavidin (Tr) by FtsK after 180 s was determined by gel electrophoresis, with fluorescent visualization of DNA. FtsK does not remain bound to DNA upon electrophoresis, but bound streptavidin or traptavidin causes the gel shift indicated with arrows. Controls are shown without streptavidin or traptavidin or without ATP, preventing FtsK activity. The percentage of free DNA and the percentage of DNA displaced by FtsK for duplicate assays are indicated under each lane. (d) Streptavidin is displaced by FtsK on the second time-scale. Streptavidin and traptavidin were incubated with FtsK for the indicated times and analyzed as in (c). (e) Traptavidin is displaced by a high concentration of FtsK. Streptavidin and traptavidin were incubated for 180 s at the indicated concentration of FtsK and analyzed as in (c).