Table 1.
Structure determination statistics for A/A2-1 [PDB: 4BCS].
Figure 1.
A. Cartoon representation of the homotetramer. Subunits I–IV are numbered according to Livnah et al. (1993) [42]: I, blue; II, green; III, light grey; IV, brown. B. Cartoon models of the superimposed A/A2-1 (blue) and chicken AVD (brown; [PDB:1AVD]) subunits I. C. Cartoon models of the superimposed A/A2-1 (blue) and AVR2 (brown; [PDB:1WBI]) subunits I. A–C. The bound biotin ligands are drawn as spheres; carbon atoms are shown in white, sulfur atoms in yellow, nitrogen atoms in blue and oxygen atoms in red. L1,2, the loop between β-strand 1 and 2; L2,3, the loop between β-strand 2 and 3, etc.
Figure 2.
A. A weighted 2FO-FC contour map (sigma level 1) showing electron density around biotin (BTN) and D12. The putative hydrogen bond is indicated by a dashed line. B. Salt-bridge between D39 and R112 in the A/A2-1 structure. The side chains of T75, L97 and S99 in the close vicinity of the salt bridge are also shown as stick models; oxygen atoms are shown in red and nitrogen atoms in blue. C. The salt bridge cannot form in the chicken AVD structure [PDB:1AVD] between residues A39 and R114 equivalent to residues D39 and R112 of A/A2-1. Residues T77, L99 and S101 equivalent to T75, L97 and S99 of A/A2-1 are shown. D. Salt-bridge between D39 and R112 in the AVR2 structure [PDB:1WBI]. Residues S75, Q97 and L99 equivalent to T75, L97 and S99 of A/A2-1 are shown. B–D. Cartoon models: subunit I, blue; subunit II green. Biotin (BTN) molecules are shown as spheres; coloring as in Figure 1.
Figure 3.
Sequence alignment of AVD, A/A2-1, A/A2-B and AVR2.
A/A2-1 and A/A2-B show higher similarity to AVD [NP_990651.1] than to AVR2 [NP_001025519.1]. The two mutants differ from each other only at six amino acid positions. Amino acids originating from AVD and AVR2 are respectively indicated with yellow or green background. A point mutation in A/A2-1 is indicated with pink background. The secondary structure is based on AVD [PDB: 2AVI]; the β-strands are indicated by black arrows. Residue numbering is according to AVD. Isoleucine/lysine residue (111 in avidin, 109 in AVR2, A/A2-1 and A/A2-B) is indicated by a black arrow.
Table 2.
Avidin-biotin displacement assay.
Figure 4.
Release of the fluorescence-labeled biotin probe.
The biotinylated fluorescence probe BF560 was released from the proteins at 25°C and followed over one hour after addition of free biotin. Please note the immediate release of the fluorescence probe in case of AVR2 and A/A2-1, indicating high dissociation rate.
Table 3.
Thermal transition midpoints (Tm) and the stability differences between biotin-bound and biotin-free forms (ΔTm) at different pH-values as determined by DSC.
Figure 5.
Tetramer stability of analyzed proteins as determined by SDS-PAGE stability assay.
The proteins were incubated for 20(left panel) or presence (middle and right panel) of biotin-5-fluorescein. A. AVD, B. AVR2, C. A/A2-1, D. A/A2-B, E. A/A2-B I109K. The gels were stained with Coomassie Brilliant Blue staining. The gels in the presence of biotin-5-fluorescein were imaged under UV light before staining (right panel). Numbers indicate the temperature in °C at which the samples were incubated. D: protein sample incubated at 100°C for 20 min in the presence of 2% SDS and 0.5% β-mercaptoethanol. AVD in C refers to AVD in the presence of biotin-5-fluorescein incubated at 20°C.