Continuum methods were used to calculate the electrostatic contributions of charged and polar side chains to the overall stability of a small 41-residue helical protein, the peripheral subunit-binding domain. The results of these calculations suggest several residues that are destabilizing, relative to hydrophobic isosteres. One position was chosen to test the results of these calculations. Arg8 is located on the surface of the protein in a region of positive electrostatic potential. The calculations suggest that Arg8 makes a significant, unfavorable electrostatic contribution to the overall stability. The experiments described in this paper represent the first direct experimental test of the theoretical methods, taking advantage of solid-phase peptide synthesis to incorporate approximately isosteric amino acid substitutions. Arg8 was replaced with norleucine (Nle), an amino acid that is hydrophobic and approximately isosteric, or with alpha-amino adipic acid (Aad), which is also approximately isosteric but oppositely charged. In this manner, it is possible to isolate electrostatic interactions from the effects of hydrophobic and van der Waals interactions. Both Arg8Nle and Arg8Aad are more thermostable than the wild-type sequence, testifying to the validity of the calculations. These replacements led to stability increases at 52.6 degrees C, the T(m) of the wild-type, of 0.86 and 1.08 kcal mol(-)(1), respectively. The stability of Arg8Nle is particularly interesting as a rare case in which replacement of a surface charge with a hydrophobic residue leads to an increase in the stability of the protein. Study holds ProTherm entries: 8036 Extra Details: peripheral subunit-binding domain; electrostatic potential;,isosteric; surface charge
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:35 p.m.
|Number of data points||2|
|Proteins||Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex ; Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence VIAMPSVRKYAREKGVDIRLVQGTGKNGRVLKEDIDAFLAGGA|