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Ligand binding is the principal determinant of stability for the p21(H)-ras protein
1Department of Chemistry and Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
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.
Insights
Ligands like GDP and Mg2+ are crucial for the structural stability and specific tertiary structure of the p21(H-ras) protein, a key molecular switch in cell signaling. Without these ligands, the protein maintains secondary structure but loses its functional three-dimensional shape.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Structure and Dynamics
Background:
- p21(H-ras) is a GTPase superfamily protein functioning as a molecular switch in signal transduction.
- GDP and Mg2+ are essential for maintaining the inactive conformation of p21(H-ras).
- Understanding ligand influence on protein structure and stability is critical for deciphering cellular signaling mechanisms.
Purpose of the Study:
- To investigate the impact of GDP and Mg2+ on the structural stability and unfolding transitions of p21(H-ras).
- To compare the structural properties of ternary (p21.GDP.Mg2+), binary (p21.GDP), and apo (p21) forms of the protein.
Main Methods:
- Urea-induced equilibrium unfolding monitored by absorbance and circular dichroism spectroscopies.
- Analysis of secondary and tertiary structure changes using near- and far-UV circular dichroism.
- Two-state model fitting to describe unfolding transitions and thermodynamic analysis.
Main Results:
- All three forms of p21(H-ras) exhibited cooperative unfolding described by a two-state model.
- Free energy of folding decreased significantly from ternary (14.1 kcal mol-1) to binary (7.5 kcal mol-1) to apo (1.8 kcal mol-1) forms.
- Removal of Mg2+ loosened aromatic side chain packing, while removal of both GDP and Mg2+ fully released side chain packing but preserved secondary structure.
Conclusions:
- Ligands GDP and Mg2+ play a significant role in the stability and tertiary structure formation of p21(H-ras).
- The amino acid sequence alone directs the formation of secondary structure, resembling a molten globule state.
- Ligand binding is essential for driving the specific tertiary structure formation required for p21(H-ras) function.