A novel chimeric avidin with increased thermal stability using DNA shuffling

Abstract

Avidins are a family of proteins widely employed in biotechnology. We have previously shown that functional chimeric mutant proteins can be created from avidin and avidin-related protein 2 using a methodology combining random mutagenesis by recombination and selection by a tailored biopanning protocol (phage display). Here, we report the crystal structure of one of the previously selected and characterized chimeric avidin forms, A/A2-1. The structure was solved at 1.8 Å resolution and revealed that the protein fold was not affected by the shuffled sequences. The structure also supports the previously observed physicochemical properties of the mutant. Furthermore, we improved the selection and screening methodology to select for chimeric avidins with slower dissociation rate from biotin than were selected earlier. This resulted in the chimeric mutant A/A2-B, which showed increased thermal stability as compared to A/A2-1 and the parental proteins. The increased stability was especially evident at conditions of extreme pH as characterized using differential scanning calorimetry. In addition, amino acid sequence and structural comparison of the chimeric mutants and the parental proteins led to the rational design of A/A2-B I109K. This mutation further decreased the dissociation rate from biotin and yielded an increase in the thermal stability.

Figure 1. X-ray structure of A/A2-1.

A. Cartoon representation of the homotetramer. Subunits I–IV are numbered according to Livnah et al. (1993) : 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. Unique features of A/A2-1.

A. A weighted 2F_O-F_C 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.

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.

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.