3.3 Spectroscopic, chromatographic and PET-binding characteristics of mutants.
Tf C is an α/β hydrolase which shows a CD spectrum typical of proteins with mixed α/β structures, with a spectral shape dominated by negative bands at 208 and 222 nm, and spectral intensity characterized by substantially lower MRE than is associated with α-helices. CD spectra of all mutants/variants overlapped substantially with that ofTf C, demonstrating that the enzyme tolerated the mutations, and remained folded in a native-like structural format (Figure 3A). Size exclusion chromatographic behaviour of all mutants/variants was identical to that of Tf C, with elution occurring at the same volume (~13.5 ml) from the column used (data not shown for all mutants in Figure 3B). Only L90F eluted at a later volume (~14.1 ml), due to hydrophobic interactions with the column matrix, owing to the enhanced surface hydrophobicity (Figure 3B).
We performed PET-binding assays, based on SDS-PAGE analyses of enzyme populations seen in lanes corresponding to reaction supernatants, or extracts of PET-bound populations (Figure 3C), followed by densitometric analyses (Figure 3D). These assays were performed for mutants/variants, G62A/L90F, G62A/F209I and G62A/F249R. Binding of L90F to PET was noted to be comparable to that of Tf C, in that the partitioning of enzyme populations between PET (lane 1 versus lane 3) and solution (lane 2 versus lane 4) were similar. In contrast, with the G62A/F209I variant, significantly less enzyme was seen to have remained bound to PET (lane 6 and Figure 3D), with proportionate increase in the enzyme left in solution (lane 7). Figure 3D shows that PET binding by G62A/F249R is intermediate to that of G62A/L90F and G62A/F209I. The differences would appear to owe to differences in overall surface hydrophobicity, wrought through the protein engineering carried out.