Stability of monovalent streptavidin. (a) Stability to subunit exchange was determined by incubating 5 μM A1D3 in PBS as indicated, and detecting rearranged tetramers by 8% SDS-PAGE, by comparison to the initial product of refolding of D and A in a 3:1 ratio (Mix). (b) Stability of tetramer to heat denaturation was determined by incubating 5 μM wild-type streptavidin or A1D3 in PBS at the indicated temperatures for 3 min and then analyzing on 16% SDS-PAGE. M, marker.

Effect of monovalent and wild-type streptavidin on neuroligin-1 clustering. (a) Hippocampal neurons in dissociated culture were transfected with AP–neuroligin-1, biotinylated with biotin ligase and labeled with Alexa Fluor 568–conjugated wild-type (left) or A1D3 (right) streptavidin. After staining, cells were incubated for 0h (top) or 2 h (middle) at 37 °C and streptavidin staining was visualized by live-cell fluorescence microscopy. Scale bar, 10 μm. Magnified images of the boxed regions are shown at the bottom. Scale bar, 1 μm. (b) Neurons were biotinylated and labeled with wild-type or A1D3 streptavidin as above, incubated for 24 h and then stained for the presynaptic marker VGLUT1. Streptavidin (red) and VGLUT1 (green) signals are shown separately or overlaid. Scale bar, 10 μm. Magnified images of the boxed regions are shown below. Scale bar, 1 μm. Arrows indicate AP–neuroligin-1 clusters not apposed to presynaptic terminals.

Generation of monovalent streptavidin. (a) Wild-type streptavidin is a tetramer with four biotin binding sites (B, biotin). Monovalent streptavidin is a tetramer with 3 inactive subunits (dark gray) and one subunit that binds biotin with wild-type affinity (light gray). (b) Biotin binding site of wild-type streptavidin (from Protein Data Bank 1MK5)11, highlighting the three residues mutated to create the ‘dead’ subunit (left). Asn23 and Ser45 were changed to alanines, removing two hydrogen bonds (dashed lines) to biotin, and Ser27 was changed to aspartate, to introduce a steric clash. In the monovalent streptavidin, the biotin binding site near the subunit interface and the residues mutated in the dead subunits are shown in green (right). (c) To make monovalent streptavidin, dead streptavidin subunits (D) and wild-type streptavidin subunits (A) in a 3:1 ratio were refolded from denaturant, giving a mix of streptavidin heterotetramers. Tetramers with a single 6His-tagged wild-type subunit were purified on a Ni-NTA column. (d) SDS-PAGE of chimeric streptavidins under nondenaturing conditions. Streptavidin with 4 dead subunits (D4), wild-type streptavidin with a 6His-tag (A4), the product of refolding of D and A in a 3:1 ratio (Mix), and chimeric tetramers with one (A1D3), two (A2D2) or three (A3D1) biotin binding subunits were loaded without boiling onto a polyacrylamide gel and visualized by Coomassie staining. (e) SDS-PAGE of chimeric streptavidins under denaturing conditions to break the tetramer into monomers.

Affinity and off rate of biotin binding to chimeric tetramers. (a) To determine the K_d for D4, 24 μM D4 was incubated with increasing concentrations of [³H]biotin. After 20 h, the amount of bound [³H]biotin was determined by precipitating D4. Means of triplicate measurements are shown ±1 s.d. Some error bars are too small to be visible. (b) To determine the K_d for A1D3, A2D2 and A4, increasing concentrations of A1D3, A2D2 or A4 were incubated with 20 nM [³H]biotin and 50 nM wild-type streptavidin. After 20 h, chimeric tetramers were removed using Ni-NTA agarose, and the amount of [³H]biotin bound to wild-type streptavidin in the supernatant was measured. From this value, the amount of [³H]biotin bound to the chimeric tetramers was deduced. Means of triplicate measurements are shown ±1 s.d. (c) Off rate from a biotin conjugate. Wild-type, A1D3, S45A or T90I streptavidin was added in excess to biotin-4-fluorescein to quench its fluorescence. Excess competing biotin was added and fluorescence increase was monitored as biotin-4-fluorescein dissociated from streptavidin. The 100% value represents complete dissociation of biotin-4-fluorescein. Means of triplicate measurements are shown +1 s.d. The bottom panel is a magnification of the 0–10% region of the y axis, to illustrate the similar dissociation curves for wild-type streptavidin and A1D3. (d) Off rate from biotin. A1D3 or wild-type streptavidin was incubated with [³H]biotin. Excess cold biotin was then added. After varying amounts of time at 37 °C, the amount of bound [³H]biotin was determined by precipitating streptavidin. Means of triplicate measurement are shown ±1 s.d.