Changes in stability upon charge reversal and neutralization substitution in staphylococcal nuclease are dominated by favorable electrostatic effects.


Abstract

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. Study holds ProTherm entries: 15907, 15908, 15909, 15910, 15911, 15912, 15913, 15914, 15915, 15916, 15917, 15918, 15919, 15920, 15921, 15922, 15923, 15924, 15925, 15926, 15927, 15928, 15929, 15930, 15931, 15932, 15933, 15934, 15935, 15936, 15937, 15938, 15939, 15940, 15941, 15942, 15943, 15944, 15945, 15946, 15947, 15948, 15949 Extra Details: surface charge; alanine; electrostatic free energy; continuum method; dielectric constant; dynamic fluctuations

Submission Details

ID: 8BKSZHCa

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Schwehm JM;Fitch CA;Dang BN;GarcĂ­a-Moreno E B;Stites WE,Biochemistry (2003) Changes in stability upon charge reversal and neutralization substitution in staphylococcal nuclease are dominated by favorable electrostatic effects. PMID:12549934
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