title: Designed protein reveals structural determinants of extreme kinetic stability.
abstract: The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge.
authors.affiliation: Department of Chemistry, Guelph-Waterloo Centre for Graduate Studies in Chemistry and Biochemistry, University of Waterloo, Waterloo, ON, Canada N2L 3G1;
authors.forename: Aron
authors.initials: A
authors.lastname: Broom
authors.affiliation: Department of Chemistry, Guelph-Waterloo Centre for Graduate Studies in Chemistry and Biochemistry, University of Waterloo, Waterloo, ON, Canada N2L 3G1;
authors.forename: S Martha
authors.initials: SM
authors.lastname: Ma
authors.affiliation: Department of Chemistry and Chemical Biology, and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180;
authors.forename: Ke
authors.initials: K
authors.lastname: Xia
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore-560065, India; Manipal University, Madhav Nagar, Manipal 576104, India.
authors.forename: Hitesh
authors.initials: H
authors.lastname: Rafalia
authors.affiliation: Department of Chemistry, Guelph-Waterloo Centre for Graduate Studies in Chemistry and Biochemistry, University of Waterloo, Waterloo, ON, Canada N2L 3G1;
authors.forename: Kyle
authors.initials: K
authors.lastname: Trainor
authors.affiliation: Department of Chemistry and Chemical Biology, and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180;
authors.forename: Wilfredo
authors.initials: W
authors.lastname: Colón
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore-560065, India;
authors.forename: Shachi
authors.initials: S
authors.lastname: Gosavi
authors.affiliation: Department of Chemistry, Guelph-Waterloo Centre for Graduate Studies in Chemistry and Biochemistry, University of Waterloo, Waterloo, ON, Canada N2L 3G1; meiering@uwaterloo.ca.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Meiering
mesh_terms: D001665:Binding Sites; D003198:Computer Simulation; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D003902:Detergents; D008024:Ligands; D011506:Proteins; D010447:Peptide Hydrolases
keywords: SDS/protease resistance; coarse-grained simulations; contact order; protein folding; protein topology