Increasing protein stability using a rational approach combining sequence homology and structural alignment: Stabilizing the WW domain

This study shows that a combination of sequence homology and structural inf ormation can be used to increase the stability of the WW domain by 2.5 kcal mol(-1) and increase the T-m by 28 degreesC, Previous homology-based prote in design efforts typically investigate positions with low sequence identit y, whereas this study focuses on semi-conserved core residues and proximal residues, exploring their role(s) in mediating stabilizing interactions on the basis of structural considerations. The A20R and L30Y mutations allow i ncreased hydrophobic interactions because of complimentary surfaces and an electrostatic interaction with a third residue adjacent to the ligand-bindi ng hydrophobic cluster, increasing stability significantly beyond what addi tivity would predict for the single mutations. The D34T mutation situated i n a pi -turn possibly disengages Asn31, allowing it to make up to three hyd rogen bonds with the backbone in strand 1 and loop 2. The synergistic mutat ions A20R/L30Y in combination with the remotely located mutation D34T add t ogether to create a hYap WW domain that is significantly more stable than a ny of the protein structures on which the design was based (Pin and FBP28 W W domains).