Ligand binding is the principal determinant of stability for the p21(H)-ras protein.
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.
Department of Chemistry and Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
J
J
Zhang
C R
CR
Matthews
D001059:Apoproteins / Q000737:chemistry; D002942:Circular Dichroism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry*; D013816:Thermodynamics; D014508:Urea
D001059:Apoproteins; D008024:Ligands; D006153:Guanosine Diphosphate; D014508:Urea; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D008274:Magnesium