title: Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein.
abstract: Proteins from organisms that have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single-molecule force spectroscopy (SMFS), and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal β-strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (ΔGD-N) of the mutated proteins and show that there is a more significant reduction for TmCSP L40A (ΔΔG = 63%) than TmCSP V62A (ΔΔG = 47%), as might be expected on the basis of the relative reduction in the size of the side chain. By contrast, SMFS measures the mechanical stability (ΔG*) and shows a greater reduction for TmCSP V62A (ΔΔG* = 8.4%) than TmCSP L40A (ΔΔG* = 2.5%). While the impact on the mechanical stability is subtle, the results demonstrate the power of tuning noncovalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provide the opportunity to design proteins with optimized thermodynamic and mechanical properties.
authors.affiliation: School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, United Kingdom.
authors.forename: Katarzyna M
authors.initials: KM
authors.lastname: Tych
authors.affiliation: School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Batchelor
authors.affiliation: School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, United Kingdom.
authors.forename: Toni
authors.initials: T
authors.lastname: Hoffmann
authors.affiliation: School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, United Kingdom.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Wilson
authors.affiliation: School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, United Kingdom.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Hughes
authors.affiliation: School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, United Kingdom.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Paci
authors.affiliation: School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Brockwell
authors.affiliation: School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, United Kingdom.
authors.forename: Lorna
authors.initials: L
authors.lastname: Dougan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D000072417:Protein Domains; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins