title: Quantifying the kinetic stability of hyperstable proteins via time-dependent SDS trapping.
abstract: Globular proteins are usually in equilibrium with unfolded conformations, whereas kinetically stable proteins (KSPs) are conformationally trapped by their high unfolding transition state energy. Kinetic stability (KS) could allow proteins to maintain their activity under harsh conditions, increase a protein's half-life, or protect against misfolding-aggregation. Here we show the development of a simple method for quantifying a protein's KS that involves incubating a protein in SDS at high temperature as a function of time, running the unheated samples on SDS-PAGE, and quantifying the bands to determine the time-dependent loss of a protein's SDS resistance. Six diverse proteins, including two monomer, two dimers, and two tetramers, were studied by this method, and the kinetics of the loss of SDS resistance correlated linearly with their unfolding rate determined by circular dichroism. These results imply that the mechanism by which SDS denatures proteins involves conformational trapping, with a trapping rate that is determined and limited by the rate of protein unfolding. We applied the SDS trapping of proteins (S-TraP) method to superoxide dismutase (SOD) and transthyretin (TTR), which are highly KSPs with native unfolding rates that are difficult to measure by conventional spectroscopic methods. A combination of S-TraP experiments between 75 and 90 °C combined with Eyring plot analysis yielded an unfolding half-life of 70 ± 37 and 18 ± 6 days at 37 °C for SOD and TTR, respectively. The S-TraP method shown here is extremely accessible, sample-efficient, cost-effective, compatible with impure or complex samples, and will be useful for exploring the biological and pathological roles of kinetic stability.
authors: Department of Chemistry and Chemical Biology, and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, United States.
authors: Ke
authors: K
authors: Xia
authors: Songjie
authors: S
authors: Zhang
authors: Brendan
authors: B
authors: Bathrick
authors: Shuangqi
authors: S
authors: Liu
authors: Yeidaliz
authors: Y
authors: Garcia
authors: Wilfredo
authors: W
authors: Colón
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001963:Bromelains / Q000737:chemistry; D002374:Catalase / Q000737:chemistry; D002417:Cattle; D044602:Cellulases / Q000737:chemistry; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry; D005949:Glucose Oxidase / Q000737:chemistry; D006801:Humans; D010940:Plant Proteins / Q000737:chemistry; D011228:Prealbumin / Q000737:chemistry; D011489:Protein Denaturation; D055550:Protein Stability / Q000187:drug effects*; D058767:Protein Unfolding / Q000187:drug effects; D012967:Sodium Dodecyl Sulfate; D019097:Spin Trapping / Q000379:methods; D019809:Streptavidin / Q000737:chemistry; D013816:Thermodynamics; D013997:Time Factors; D014361:Trypsin Inhibitors / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D010940:Plant Proteins; D011228:Prealbumin; D014361:Trypsin Inhibitors; D012967:Sodium Dodecyl Sulfate; D001963:Bromelains; D019809:Streptavidin; D005949:Glucose Oxidase; D002374:Catalase; D044602:Cellulases