Protein engineering is done to increase the kinetics and stability of enzmyes. The technique involves the mutation residues until the desired phenotype is obtained. Saturation mutatgenesis and degenerate codons introduce random mutations into the parent enzyme. These "libraries" are then subjected to highthroughput phenotype screens/selections which allow for millions of mutants to be examined simultaneously. Phylogenetic analysis helps direct researchers in choosing which residues to mutatgenize allowing for more focused libraries.
Fig 1. Sites MMP3, 7, and 12 cleave IgG: located in the hinge region of the Fc region, (1) MMP7 cuts IgG at a different location than (2) MMP3 and 12. Which residues are responsible for this shift and does it affect the rate of cleavage. (3) IdeS is a therapeutic bacterial protease with high activity for IgG.
In Dr. Iverson's lab, we are interested engineering a Human Matrix Metalloprotease (MMP) for high activity for Immunoglobulin G (IgG) and low activity for elastin. MMP3, 7, and 12 are all known to cleave IgG and elastin. Suprisingly, the ratio of elastin to IgG cleavage rate for each of these enzmyes is about the same (MMP7< MMP3 < MMP12). In addition to being slower than MMP3 and MMP12, MMP7 cleaves at a different site in the IgG (Fig. 1). What causes this shift in the cleavage site and does it affect the rate of IgG cleavage?
The backbones of these three MMPs are very similar (Fig 2.). This indicates the physical characteristics of the active site residues as opposed to distal residues restructuring the active site is responsible for the differences in activity. Herein is a discussion of possible residues responsible for the increased rate of hydrolysis MMP3 and 12 exhibits for Immunoglobulin G and elastin.
Figure 2. Alpha carbon alignment of MMP3&7 and MMP7&12 respectively: Despite the differences in activies the backbone of all three enzmyes are very similar.