Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface

Biochemistry. 1999 Dec 14;38(50):16419-23. doi: 10.1021/bi992271w.


A simple theoretical model for increasing the protein stability by adequately redesigning the distribution of charged residues on the surface of the native protein was tested experimentally. Using the molecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surface of this protein which would lead to an increase in its stability. Experimental validation for this prediction was achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-induced unfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin are indeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. As a positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of the ubiquitin molecule were considered as well. These predictions were also tested experimentally using correspondingly designed variants of ubiquitin. We found that these variants are less stable than the wild-type protein, again in agreement with the theoretical prediction. These observations provide guidelines for rational design of more stable proteins and suggest a possible mechanism of structural stability of proteins from thermophilic organisms.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution / genetics
  • Arginine / chemistry
  • Arginine / genetics
  • Asparagine / chemistry
  • Asparagine / genetics
  • Circular Dichroism
  • Glutamic Acid / chemistry
  • Glutamic Acid / genetics
  • Glutamine / chemistry
  • Glutamine / genetics
  • Histidine / chemistry
  • Histidine / genetics
  • Linear Models
  • Lysine / chemistry
  • Lysine / genetics
  • Mutagenesis, Site-Directed
  • Protein Engineering*
  • Protein Folding
  • Static Electricity
  • Surface Properties
  • Thermodynamics
  • Ubiquitins / chemistry*
  • Ubiquitins / genetics*


  • Ubiquitins
  • Glutamine
  • Glutamic Acid
  • Histidine
  • Asparagine
  • Arginine
  • Lysine