title: High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering.
abstract: The low stability of natural proteins often limits their use in therapeutic, industrial, and research applications. The scale and throughput of methods such as circular dichroism, fluorescence spectroscopy, and calorimetry severely limit the number of variants that can be examined. Here we demonstrate a high-throughput thermal scanning (HTTS) method for determining the approximate stabilities of protein variants at high throughput and low cost. The method is based on binding to a hydrophobic dye akin to ANS, which fluoresces upon binding to molten globules and thermal denaturation intermediates. No inherent properties of the protein, such as enzymatic activity or presence of an intrinsic fluorophore, are required. Very small sample sizes are analyzed using a real-time PCR machine, enabling the use of high-throughput purification. We show that the apparent T(M) values obtained from HTTS are approximately linearly related to those from CD thermal denaturation for a series of four-helix bundle hydrophobic core variants. We demonstrate similar results for a small set of TIM barrel variants. This inexpensive, general, and scaleable approach enables the search for conservative, stable mutants of biotechnologically important proteins and provides a method for statistical correlation of sequence-stability relationships.
authors.affiliation: Ohio State Biochemistry Program, Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Lavinder
authors.forename: Sanjay B
authors.initials: SB
authors.lastname: Hari
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Sullivan
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Magliery
mesh_terms: D005456:Fluorescent Dyes; D008722:Methods; D015336:Molecular Probe Techniques; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry; D044366:Transition Temperature
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins