A series of highly conserved flanking cysteines (Cys-53, Cys-77, and Cys-83) were mutated to alanine, but none of these mutations showed any effect on either enzyme activity.
For M5B, 8 bp from 33 to 40 were mutated to (5'-GAATTCTG-3'); for M6B, 12 bp from 41 to 52 were mutated to "TGGTTGAATTCT"; for M7B, 12 bp from 53 to 64 were mutated to 5'-TGTGAATTCGTG-3'; for M8B, 12 bp from 65 to 76 were mutated to (5'-TTGGAATTCGGG-3'); for M9B, 9 bp from 77 to 85 were mutated to (5'-GTGAATTCG-3'); for M10B, 11 bp from 86 to 96 were mutated to (5'-GAATTCTGTTG-3').
We also made a double mutation N316AF320A, in which both Asn316 and Phe320 were mutated to Ala.
Three amino acids were mutated to express immunogenic epitopes from other circulating H5N1s in the backbone.
Therefore, W120, L124 and V125 were mutated to hydrophilic residues to increase dimer solubility.
The C terminus was truncated after Ser407, and three cysteine residues were mutated to alanine (Cys396Ala, Cys400Ala, and Cys405Ala).
Second, the five cysteine residues of telethonin were mutated to serine to avoid severe problems with cysteine oxidation.
To further investigate the role of the C2HC ZnF domain, two of the predicted Zn2+ chelating residues, C100 and C103, were mutated to alanines (Figs 1D and 3A).
Mutants were also generated in which At4g18010-P3 BRRE and At4g00360-P3 E-box were mutated to unrelated sequences (see Fig. 5a).
In the engineered form of SCD, lysyl residues at positions 33, 35, and 36 were mutated to alanine (SCD K/A).
In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57VL85S and FV57VL84S.
