title: Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins.
abstract: The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H(+) binding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pK(a) values in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pK(a) values in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pK(a) values of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking.
authors.affiliation: Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, United States.
authors.forename: Peregrine
authors.initials: P
authors.lastname: Bell-Upp
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Robinson
authors.forename: Steven T
authors.initials: ST
authors.lastname: Whitten
authors.forename: Erika L
authors.initials: EL
authors.lastname: Wheeler
authors.forename: Janine
authors.initials: J
authors.lastname: Lin
authors.forename: Wesley E
authors.initials: WE
authors.lastname: Stites
authors.forename: Bertrand García-Moreno
authors.initials: BG
authors.lastname: E
mesh_terms: D000143:Acids / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D055550:Protein Stability; D058767:Protein Unfolding; D013210:Staphylococcus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics
chemical_list: D000143:Acids; D008836:Micrococcal Nuclease