Changes in Stability upon Charge Reversal and Neutralization Substitution in Staphylococcal Nuclease Are Dominated by Favorable Electrostatic Effects

• Published in: Biochemistry (Easton) 2003-02, Vol.42 (4), p.1118-1128
• Main Authors: Schwehm, Jeffery M, Fitch, Carolyn A, Dang, Bao N, García-Moreno E, Bertrand, Stites, Wesley E
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Aspartic Acid - genetics; Enzyme Stability - genetics; Glutamic Acid - genetics; Glutamine - genetics; Guanidine - chemistry; Histidine - genetics; Hydrogen-Ion Concentration; Lysine - genetics; Micrococcal Nuclease - chemistry; Micrococcal Nuclease - genetics; Models, Chemical; Normal Distribution; Poisson Distribution; Protein Denaturation; Protein Folding; Static Electricity; Surface Properties; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0266434

Single site mutations that reverse or neutralize a surface charge were made at 22 ionizable residues in staphylococcal nuclease. Unfolding free energies were obtained by guanidine hydrochloride denaturation. These data, in conjunction with previously obtained stabilities of the corresponding alanine mutants, unequivocally show that the dominant contribution to stability for virtually all of the wild-type side chains examined is the electrostatic effect associated with each residue's charged group. With only a few exceptions, these charges stabilize the native state, with an average loss of 0.5 kcal/mol of stability upon neutralization of a charge. When the charge is reversed, the average destabilization is doubled. Structure-based calculations of electrostatic free energy with the continuum method based on the finite difference solution to the linearized Poisson−Boltzmann equation reproduce the observed energetics when the polarizability in the protein interior is represented with a dielectric constant of 20. However, in some cases, large differences are found, giving insight into possible areas for improvement of the calculations. In particular, it appears that the assumptions made in the calculations about the absence of electrostatic interactions in the denatured state and the energetic consequences of dynamic fluctuations in the native state will have to be further explored.