Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles

Abstract

Coulombic interactions between charges on the surface of proteins contribute to stability. It is difficult, however, to estimate their importance by protein engineering methods because mutation of one residue in an ion pair alters the energetics of many interactions in addition to the coulombic energy between the two components. We have estimated the interaction energy between two charged residues, Asp-12 and Arg-16, in an alpha-helix on the surface of a barnase mutant by invoking a double-mutant cycle involving wild-type enzyme (Asp-12, Thr-16), the single mutants Thr----Arg-16 and Asp----Ala-12, and the double mutant Asp----Ala-12, Thr----Arg-16. The changes in free energy of unfolding of the single mutants are not additive because of the coulombic interaction energy. Additivity is restored at high concentrations of salt that shield electrostatic interactions. The geometry of the ion pair in the mutant was assumed to be the same as that in the highly homologous ribonuclease from Bacillus intermedius, binase, which has Asp-12 and Arg-16 in the native enzyme. The ion pair does not form a hydrogen-bonded salt bridge, but the charges are separated by 5-6 A. The mutant barnase containing the ion pair Asp-12/Arg-16 is more stable than wild type by 0.5 kcal/mol, but only a part of the increased stability is attributable to the electrostatic interaction. We present a formal analysis of how double-mutant cycles can be used to measure the energetics of pairwise interactions.

Protein folding and stability: the pathway of folding of barnase

“…Particular interactions between two residues in the protein may, however, frequently be separated from the rest as follows [33]. Two residues, X and Y, in a protein E that interact are mutated separately and then together to give a cycle comprising E-XY, EX, EY and E. In ideal circumstances, where the mutations cause no rearrangement in the protein structure, subtraction of the free energy change AGE_X+E (for the process of E-X+E) from A%XY-,EY (the free energy change for E-XY+EY) leads to the interaction energies of X and Y with the rest of the protein to cancel out [33,34]. This still holds if there is disruption of the structure of the enzyme on mutation of X in E-XY but the same disruption occurs on the mutation of X in E-X.…”

Effects of salt bridges on protein structure and design

“…Studies of "compound" and "cooperative" interactions have expanded our understanding beyond simply excluded volume, dispersion interactions, the hydrophobic effect (Kauzmann, 1959), hydrogen bonds (Baker & Hubbard, 1984;Fersht et al, 1985;Yang et al, 1992), salt bridges (Fersht, 1971(Fersht, , 1972Marqusee & Baldwin, 1987;Anderson et al, 1990;Horovitz et al, 1990;Serrano et al, 1990;Dao-pin et al, 1991;Merutka & Stellwagen, 1991;Lyu et al, 1992;HuyghuesDespointes et al, 1993;Hendsch & Tidor, 1994;Waldburger et al, 1995;Wimley et al, 1996) and local backbone and side-chain entropy (Page & Jencks, 1971;Matthews et al, 1987;Straatsma & McCammon, 1989;Tobias et al, 1989;Pickett & Sternberg, 1993) to include more aggregate and delocalized effects. Examples range from the medium-ranged, such as helix-capping interactions (Hol et al, 1978;Sheridan et al, 1982;Hol, 1985;Shoemaker et al, 1987;Nicholson et al, 1988Nicholson et al, , 1991Presta & Rose, 1988;Richardson & Richardson, 1988;Sali et al, 1988;Fairman et al, 1989;Serrano & Fersht, 1989;Lyu et al, 1993;Hendsch & Tidor, 1994;Tidor, 1994;PrCvost, 1996) to the global, such as overall vibrational entropy …”

A few amino acid substitutions are responsible for the higher thermostability of a novel NAD+‐dependent bacillar alcohol dehydrogenase

“…Lysll is a highly conserved residue in the compared maize (ADH1, Lys20), horse liver (ADH E subunit, Lysl9), human (ADH j3l subunit, Lys20) as well as other bacillar ADH [22]. Thus, the charge substitution due to a glutamic acid replacing a lysine and the introduction of a lysine in position 14 might contribute to the higher thermostability of ADH-hT through the generation of an additional surface salt bridge [21], which increases the helix-forming potential of the N-terminal region, as previously demonstrated both for some amino acid stretches of proteins [23] and for synthetic small peptides in solution [24]. Likewise, proline imposes rigidity to polypeptide chains particularly when it substitutes for small amino acid residues such as glycine and alanine in loops between elements of secondary structure [25, 261. Therefore Pro242 can stabilize the native form of ADH-hT just by exploiting a change in the peptide backbone which contributes to an overall decrease in the entropy of the unfolded state.…”