title: A single disulfide bond restores thermodynamic and proteolytic stability to an extensively mutated protein.
abstract: The potential for engineering stable proteins with multiple amino acid substitutions was explored. Eleven lysine, five methionine, two tryptophan, one glycine, and three threonine substitutions were simultaneously made in barley chymotrypsin inhibitor-2 (CI-2) to substantially improve the essential amino acid content of the protein. These substitutions were chosen based on the three-dimensional structure of CI-2 and an alignment of homologous sequences. The initial engineered protein folded into a wild-type-like structure, but had a free energy of unfolding of only 2.2 kcal/mol, considerably less than the wild-type value of 7.5 kcal/mol. Restoration of the lysine mutation at position 67 to the wild-type arginine increased the free energy of unfolding to 3.1 kcal/mol. Subsequent cysteine substitutions at positions 22 and 82 resulted in disulfide bond formation and a protein with nearly wild-type thermodynamic stability (7.0 kcal/mol). None of the engineered proteins retained inhibitory activity against chymotrypsin or elastase, and all had substantially reduced inhibitory activity against subtilisin. The proteolytic stabilities of the proteins correlated with their thermodynamic stabilities. Reduction of the disulfide bond resulted in substantial loss of both thermodynamic and proteolytic stabilities, confirming that the disulfide bond, and not merely the cysteine substitutions, was responsible for the increased stability. We conclude that it is possible to replace over a third of the residues in CI-2 with minimal disruption of stability and structural integrity.
authors.affiliation: Pioneer Hi-Bred International, Inc., Johnston, Iowa 50131-1004, USA.
authors.forename: K R
authors.initials: KR
authors.lastname: Roesler
authors.forename: A G
authors.initials: AG
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D001467:Hordeum / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins; D011487:Protein Conformation; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D010455:Peptides; D010940:Plant Proteins; C093336:chymotrypsin inhibitor 2