Effect of pH on thermal- and chemical-induced denaturation of GFP

Appl Biochem Biotechnol. 2005 Aug;126(2):149-56. doi: 10.1385/abab:126:2:149.

Abstract

Green fluorescent protein (GFP) is an unusually stable autofluorescent protein that is increasingly being exploited for many applications. In this report, we have used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with sodium dodecyl sulfate (SDS), urea, and heat. Surprisingly, SDS (up to 0.5%) did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5 buffers; however, at pH 6.5, the protein lost all fluorescence within 1 min of incubation. Similarly, incubation of GFP with 8 M urea at 50 degrees C resulted in time dependent denaturation of GFP, but only in pH 6.5 buffer. At higher pH values (pH 7.5 and pH 8.5), the GFP was quite stable in 8 M urea at 50 degrees C, showing only a slight decrease in fluorescence. Heat denaturation of GFP was found to be pH dependent as well, with the denaturation being fastest at pH 6.5 as compared to pH 7.5 or pH 8.5. Like the denaturation studies, renaturation of heat-denatured GFP was most efficient at pH 8.5, followed by pH 7.5, and then pH 6.5. These results suggests that GFP undergoes a structural/stability shift between pH 6.5 and pH 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / genetics
  • Green Fluorescent Proteins / drug effects*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / isolation & purification
  • Green Fluorescent Proteins / metabolism
  • Hot Temperature*
  • Hydrogen-Ion Concentration
  • Protein Denaturation / drug effects*
  • Protein Renaturation
  • Recombinant Proteins / drug effects
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sodium Dodecyl Sulfate / pharmacology*
  • Spectrometry, Fluorescence
  • Surface-Active Agents / pharmacology*
  • Time Factors
  • Urea / pharmacology*

Substances

  • Recombinant Proteins
  • Surface-Active Agents
  • Green Fluorescent Proteins
  • Sodium Dodecyl Sulfate
  • Urea