title: Thermodynamic genetics of the folding of the B1 immunoglobulin-binding domain from streptococcal protein G.
abstract: A method has been developed to select proteins that are thermodynamically destabilized yet still folded and functional. The DNA encoding the B1 IgG-binding domain from Group G Streptococcus (Strp G) has been fused to gene III of bacteriophage M13. The resulting fusion protein is displayed on the surface of the phage thus enabling the phage to bind to IgG molecules. In addition, these phage exhibit a small plaque phenotype that is reversed by mutations that destabilize the Strp G domain. By selecting phage with large plaque morphology that retain their IgG-binding function, it is possible to identify mutants that are folded but destabilized compared with wild-type Strp G. Such mutants can be divided into three general categories: 1) those that disrupt packing of hydrophobic side chains in the protein interior; 2) those that destabilize secondary structure; and 3) those that alter specific hydrogen bonds involving amino acid side chains. A number of the mutants have been physically characterized by circular dichroism and nuclear magnetic resonance and have been shown to have structures similar to wild-type Strp G but stabilities that were decreased by 2-5 kcal/mol.
authors.affiliation: DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0328, USA.
authors.forename: K T
authors.initials: KT
authors.lastname: O'Neil
authors.forename: R H
authors.initials: RH
authors.lastname: Hoess
authors.forename: D P
authors.initials: DP
authors.lastname: Raleigh
authors.forename: W F
authors.initials: WF
authors.lastname: DeGrado
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D005822:Genetic Vectors / Q000737:chemistry; D007074:Immunoglobulin G / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011489:Protein Denaturation; D017510:Protein Folding; D013057:Spectrum Analysis; D013291:Streptococcus / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G