Vibrational structure of GDP and GTP bound to RAS: an isotope-edited FTIR study

Area of Science:

• Biochemistry
• Spectroscopy
• Structural Biology

Background:

• Vibrational spectroscopy is crucial for understanding ligand-protein interactions.
• Assigning both symmetric and antisymmetric vibrational modes is necessary for a complete description.
• Antisymmetric modes, requiring IR difference spectroscopy, have been historically inaccessible.

Purpose of the Study:

• To develop a methodology for IR difference spectroscopy to assign antisymmetric vibrational modes.
• To accurately describe ligand-protein bonding using vibrational analysis.
• To investigate bonding changes of Mg.GDP and Mg.GTP upon binding to the RAS active site.

Main Methods:

• Development of IR difference spectroscopy techniques.
• Application of Raman and IR difference spectroscopy to isotopically enriched enzyme-bound ligands.
• Analysis of vibrational mode assignments to determine structural and bonding changes.

Main Results:

• The study successfully developed and applied IR difference spectroscopy to assign antisymmetric modes.
• RAS binding alters the gamma-phosphate O--P--O angle of GTP by 2.7 degrees, with comparable angular freedom to solution.
• The beta-phosphate motion of GDP is restricted, and the beta,gamma-bridging O-P bond of GTP is slightly weakened in the active site.

Conclusions:

• The developed IR spectroscopy method enables accurate description of ligand-protein bonding.
• Binding to RAS induces specific conformational and electronic changes in GDP and GTP.
• The observed changes support a RAS-mediated hydrolysis mechanism similar to solution-phase hydrolysis.