Protein engineering the surface of enzymes

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Protein surfaces mediate interactions and are composed of charged, polar, and hydrophobic residues.
• The pH-dependent charge state of seven amino acids significantly impacts protein stability and activity.
• Titratable residues are predominantly located on the surface of soluble proteins.

Purpose of the Study:

• To analyze the residue composition and solvent exposure patterns across diverse protein families.
• To investigate the relationship between pH, protein stability, and enzymatic activity using cutinase as a model.
• To provide guidelines for optimizing protein surface properties through engineering.

Main Methods:

• Analysis of residue composition and surface exposure in 4038 proteins from 125 families.
• pH-dependent stability studies of native cutinase using differential scanning calorimetry.
• Enzymatic activity assays of cutinase with tributyrin.
• Molecular modeling of pH-dependent electrostatic potentials.
• High-resolution X-ray diffraction analysis of cutinase mutants.

Main Results:

• 40.7% of residues are significantly exposed (>30% solvent exposure), with distinct patterns for individual residue types.
• Cutinase activity onset correlates with active site histidine deprotonation, while activity loss at high pH is linked to structural destabilization.
• A negative electrostatic potential develops in the active site cleft within the enzyme's optimal activity range.

Conclusions:

• Protein surface residue composition exhibits predictable patterns relevant to protein engineering.
• Understanding pH-dependent stability and activity is crucial for enzyme optimization.
• Engineering protein surfaces can enhance stability and modulate enzymatic function.