Ligand binding is the principal determinant of stability for the p21(H)-ras protein

Biochemistry. 1998 Oct 20;37(42):14881-90. doi: 10.1021/bi9811157.

Abstract

p21(H-ras) is a 21 kDa, alpha/beta sheet protein that, as a member of the GTPase superfamily, acts as a molecular switch in signal transduction pathways. The essential role of GDP and Mg2+ in maintaining the inactive conformation of p21(H-ras) prompted a study of the influence of these ligands on its structure and stability. The urea-induced equilibrium unfolding transitions for the ternary (p21.GDP.Mg2+), binary (p21.GDP) and apo (p21) forms of p21(H-ras) at pH 7.5 and 25 degreesC were monitored by absorbance and circular dichroism spectroscopies. The cooperative disruptions of the secondary and tertiary structures for all three forms are well-described by a two-state model. Global analysis of the equilibrium unfolding data yields a free energy of folding in the absence of urea and under standard state conditions of 14.1 +/- 0.2 kcal mol-1, 7.5 +/- 0.4 kcal mol-1 and 1.8 +/- 0.2 kcal mol-1 for ternary, binary and apo forms, respectively. Near- and far-UV circular dichroism spectra of these three forms of p21(H-ras) show that removal of the Mg2+ from the ternary complex loosens the aromatic side chain packing but leaves the secondary structure largely unchanged. The removal of both GDP and Mg2+ completely releases the side chain packing but leaves a substantial fraction of the secondary structure intact. These results demonstrate that ligands play a significant role in the stability and structure of the p21.GDP.Mg2+ complex. The amino acid sequence itself only contains sufficient information to direct the formation of a large portion of the secondary structure in a molten globule-like state. Ligand binding is required to drive the formation of specific tertiary structure.

Publication types

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

MeSH terms

  • Apoproteins / chemistry
  • Circular Dichroism
  • Guanosine Diphosphate / chemistry
  • Humans
  • Ligands
  • Magnesium / chemistry
  • Models, Chemical
  • Models, Molecular
  • Protein Binding
  • Protein Folding
  • Protein Structure, Secondary
  • Proto-Oncogene Proteins p21(ras) / chemistry*
  • Thermodynamics
  • Urea

Substances

  • Apoproteins
  • Ligands
  • Guanosine Diphosphate
  • Urea
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)
  • Magnesium