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Increasing protein stability by altering long‐range coulombic interactions
Journal article   Open access   Peer reviewed

Increasing protein stability by altering long‐range coulombic interactions

Gerald R. Grimsley, Kevin L. Shaw, Lanette R. Fee, Roy W. Alston, BEATRICE M.P. Huyghues‐Despointes, Richard L. Thurlkill, J. Martin Scholtz and C. Nick Pace
Protein science, Vol.8(9), pp.1843-1849
1999
DOI: 10.1110/ps.8.9.1843
PMCID: PMC2144408
PMID: 10493585
url
https://europepmc.org/articles/pmc2144408View
Published (Version of record) Open Access

Abstract

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI ≈︁ 3.5 and a net charge of ≈︁ –6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 Å from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild‐type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild‐type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild‐type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.
 electrostatic interactions histidine pKa protein folding protein stability reverse hydrophobic effect ribonuclease Sa ribonuclease T1

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