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Increasing protein stability by altering long-range coulombic interactions

Published online by Cambridge University Press:  01 September 1999

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
GERALD R. GRIMSLEY
Affiliation:
Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843
KEVIN L. SHAW
Affiliation:
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
LANETTE R. FEE
Affiliation:
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
ROY W. ALSTON
Affiliation:
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
BEATRICE M.P. HUYGHUES-DESPOINTES
Affiliation:
Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843
RICHARD L. THURLKILL
Affiliation:
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
J. MARTIN SCHOLTZ
Affiliation:
Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843 Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 Center for Macromolecular Design, Texas A&M University, College Station, Texas 77843
C. NICK PACE
Affiliation:
Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843 Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 Center for Macromolecular Design, Texas A&M University, College Station, Texas 77843
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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.

Keywords

electrostatic interactionshistidine pKaprotein foldingprotein stabilityreverse hydrophobic effectribonuclease Saribonuclease T1

Information

Type
Research Article
Information
Protein Science , Volume 8 , Issue 9 , September 1999 , pp. 1843 - 1849
Copyright
© 1999 The Protein Society

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