Chemistry Reference
In-Depth Information
5.3.8
Scaffold Redesign
While site-directed mutagenesis for the incorporation of new functional groups into
the protein cavity is the most obvious type of genetic modification that can be per-
formed with these systems, more global changes can be made in the scaffold using
similar methods. For example, as noted above, the IFABP architecture consists of a
b
-helical lid. To examine whether entry or exit of
substrates or products into or out of this closed cavity was limiting catalytic turnover, a
helixless (hs) IFABP mutant was prepared by deleting 17 helical residues (15-31) from
the N-terminus and replacing them with a dipeptide linker (Ser-Gly) using site-direc-
ted mutagenesis [54]. A previous NMR study showed that this mutant preserved the
original
-barrel structure caped off with a
a
-helical lid and was still relatively
stable [55]. The structures of IFABP and hsIFABP are compared in Figure 5.13; except
for the absence of the
b
-sheet secondary structure without the
a
-helical lid, the overall protein fold is largely intact. Studies that
investigated reactions with this conjugate (hsIFABP-PX60) revealed that in 24 h,
approximately two turnovers were observed with a selectivity of 93% ee. A kinetics
analysis indicated that k
cat
s were comparable for both IFABP-PX60 (0.20 h
-1
) and
a
Figure 5.13 Comparison of the structures of IFABP and the helixless variant
hsIFABP. Top: Stereo view of IFABP. Bottom: Stereo view of hsIFABP.
