Figure 5. Monovalent Tr off-rate and thermostability.

(A) Tr4 (left-hand panel) is tetravalent, with each subunit binding one biotin (b). Monovalent Tr contains one Tr subunit (white square) and three D subunits (grey squares), which cannot bind biotin. Tr but not D subunits have His₆ tags (bold diagonal line). (B) Off-rate of a biotin conjugate from Tr1D3 compared with tetravalent SA and Tr4, in the presence of competing free biotin at 37 °C. Means of triplicate readings are shown ±S.D. (C) Thermostability of the monovalent traptavidin tetramer, incubated at the indicated temperature for 3 min and analysed by SDS/PAGE and Coomassie Blue staining. The positive control (c) was mixed with SDS before heating at 95 °C. Bands from tetrameric Tr1D3 or monomers (Tr or D) are indicated. The percentage monomer from Tr1D3 from duplicate gels (adjacent striped columns) is plotted against the temperature in the lower panel, with comparison with Tr4 (white columns) or D4 (filled columns).