Nucleotide hydrolysis-dependent conformational changes in p21(ras) as studied using ESR spectroscopy

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The proto-oncogene product p21(ras) plays a critical role in cellular signaling pathways.
• Understanding the structural dynamics of p21(ras) during nucleotide hydrolysis is crucial for deciphering its function.
• GTP hydrolysis by p21(ras) is a key regulatory event linked to conformational changes.

Purpose of the Study:

• To investigate structural changes in p21(ras) dependent on nucleotide hydrolysis using Electron Spin Resonance (ESR) spectroscopy.
• To characterize the conformational states of p21(ras) bound to different guanine nucleotide analogs.
• To determine the temporal relationship between GTP hydrolysis and protein conformational changes.

Main Methods:

• Utilized ESR spectroscopy with spin-labeled guanine nucleotides (SL-GTP, SL-GDP, SL-guanylylimidodiphosphate).
• Analyzed ESR spectra of p21(ras)-nucleotide complexes at different temperatures (0°C, 5°C, 25°C).
• Calculated rate constants for conformational changes and compared them with hydrolysis rates.

Main Results:

• ESR spectra of p21(ras)-SL-GTP and p21(ras)-SL-GDP complexes differed significantly, indicating distinct protein conformations.
• SL-GTP served as an effective substrate analog, exhibiting hydrolysis rates comparable to GTP.
• The rate constant for conformational change, determined by ESR, closely matched the hydrolysis rate of SL-GTP.

Conclusions:

• GTP hydrolysis by p21(ras) is accompanied by a simultaneous conformational change in the protein.
• The findings support a model where structural rearrangements occur concurrently with the enzymatic hydrolysis step.
• This study provides direct evidence linking nucleotide hydrolysis to dynamic structural alterations in p21(ras).