Abstract: Methods are provided for engineering non-naturally occurring proteins comprising artificial pH-sensitive conformational switches that respond to a change in pH by causing a global unfolding of the proteins. Non-naturally occurring proteins comprising artificial pH-sensitive conformational switches that respond to a change in pH by causing a global unfolding of the proteins are also provided.

Abstract: Internal polar and ionizable groups are essential for enzymatic catalysis, proton transport, redox reactions, and many other functional properties of proteins. To engineer novel enzymes or to modify the function of existing ones, and to build switches that can be used to modify the stability of proteins in response to changes in pH, it is necessary to introduce polar or ionizable groups or to modify the properties of existing ones. However, internal polar and ionizable groups usually destabilize proteins. The disclosure provides new methods that allow the introduction of polar and ionizable groups into the interior of proteins, as well as new methods for improving the accuracy of pKa of an internal amino acid of a protein, and methods for mapping the folding free energy landscape of a protein.

Abstract: Methods are provided for engineering non-naturally occurring proteins comprising artificial pH-sensitive conformational switches that respond to a change in pH by causing a global unfolding of the proteins. Non-naturally occurring proteins comprising artificial pH-sensitive conformational switches that respond to a change in pH by causing a global unfolding of the proteins are also provided.

Abstract: Internal polar and ionizable groups are essential for enzymatic catalysis, proton transport, redox reactions, and many other functional properties of proteins. To engineer novel enzymes or to modify the function of existing ones, and to build switches that can be used to modify the stability of proteins in response to changes in pH, it is necessary to introduce polar or ionizable groups or to modify the properties of existing ones. However, internal polar and ionizable groups usually destabilize proteins. The disclosure provides new methods that allow the introduction of polar and ionizable groups into the interior of proteins, as well as new methods for improving the accuracy of pKa of an internal amino acid of a protein, and methods for mapping the folding free energy landscape of a protein.