Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Understanding protein conformational changes is crucial for biological function.
• Existing methods for real-time detection can be limited in scope or application.
• Protein dynamics play a key role in molecular interactions and cellular processes.

Purpose of the Study:

• To develop a broadly applicable technique for real-time detection and measurement of protein conformational changes in solution.
• To demonstrate the utility of this method using well-characterized proteins and a specific mutant.
• To correlate changes in signal with specific ligand-induced structural motions.

Main Methods:

• Tethering proteins labeled with a second-harmonic generation (SHG) active dye to supported lipid bilayers.
• Measuring SHG signal changes upon ligand binding to calmodulin (CaM), maltose-binding protein (MBP), and dihydrofolate reductase (DHFR).
• Investigating structural motion at a specific site using a single-site cysteine mutant of DHFR.

Main Results:

• SHG signal changes upon ligand binding directly correlate with structural motions between apo and ligand-bound protein forms.
• Different magnitudes of SHG signal changes reflect distinct, specific ligand-induced conformational changes.
• The method successfully distinguishes between different protein conformational states.

Conclusions:

• The SHG-based approach provides a straightforward and broadly applicable method for real-time protein conformational analysis.
• This technique has significant potential for diverse biological applications requiring the study of protein dynamics.
• The method allows for sensitive detection and measurement of ligand-induced structural changes in proteins.