Solution dynamics of p21ras proteins bound with fluorescent nucleotides: a time-resolved fluorescence study

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• p21ras proteins are crucial regulators of cellular signaling pathways.
• Understanding the solution dynamics of p21ras in its GTP- and GDP-bound forms is essential for deciphering its function.
• Aberrant p21ras activity is implicated in various cancers.

Purpose of the Study:

• To investigate the solution dynamics of normal and transforming p21ras proteins.
• To differentiate the dynamics of p21ras when bound to GTP versus GDP.
• To assess the impact of specific mutations, such as Asp-12, on p21ras dynamics.

Main Methods:

• Time-resolved fluorescence spectroscopy was employed.
• Fluorescent nucleotide derivatives (mant, aminocoumarin, fluorescein) of GTP and GDP were synthesized and used as reporter groups.
• Gel permeation chromatography was used to validate findings.

Main Results:

• Fluorescence lifetimes of mant nucleotide derivatives increased upon binding to p21ras.
• A distinct difference in fluorescence lifetime was observed between normal p21ras.mantGTP and p21ras.mantGDP at 30°C, but not for the [Asp-12] mutant.
• Rotational correlation times for p21ras-bound fluorophores were significantly longer than predicted for a globular protein, indicating restricted motion.

Conclusions:

• The study provides insights into the conformational dynamics of p21ras proteins.
• Differences in dynamics are observed between normal and mutant p21ras and between GTP- and GDP-bound states.
• The observed dynamics are not artifacts of the fluorescent labels used.