Method for Incorporating Internal Polar and Ionizable Groups in Proteins

A method of stabilizing polar and ionizable groups inside of proteins, the method comprising the steps of a) increasing the thermodynamic stability of the protein;

• and b) substituting polar and ionizable groups for hydrophobic groups inside the protein, thereby stabilizing the polar and ionizable groups inside the protein.

A method of stabilizing positive and negative charges inside of proteins, the method comprising the steps of:

• a) increasing the thermodynamic stability of the protein;
• and b) substituting polar and ionizable groups for hydrophobic groups inside the protein, thereby stabilizing the positive and negative charges inside the protein.

A method of rationally modifying the thermodynamic stability of a protein as a function of pH, the method comprising the steps of:

• a) increasing the thermodynamic stability of the protein;
• and b) incorporating polar and ionizable groups for hydrophobic groups inside the protein, thereby rationally modifying the thermodynamic stability of the protein as a function of pH.

A method of engineering pH sensitive conformational switches in proteins, the method comprising the steps of:

• a) increasing the thermodynamic stability of the protein;
• and b) incorporating polar and ionizable groups for hydrophobic groups inside the protein, thereby engineering the pH sensitive conformational switch in the protein.

A method of creating novel enzymatic sites in proteins, the method comprising the steps of:

• a) increasing the thermodynamic stability of the protein;
• and b) incorporating polar and ionizable groups for hydrophobic groups inside the protein, thereby creating the novel enzymatic site in the protein.

A method for modulating the pKa of an internal amino acid of a protein, the method comprising the steps of:

• a) increasing the thermodynamic stability of the protein;
• and b) incorporating polar and ionizable groups for hydrophobic groups inside the protein, thereby modulating the pKa of the internal amino acid of the protein.

A method of mapping the folding free energy landscape of a protein, the method comprising the steps of:

• a) increasing the thermodynamic stability of the protein;
• and b) incorporating polar and ionizable groups for hydrophobic groups inside the protein, thereby mapping the folding free energy landscape of the protein.