Discovery of fragments inducing conformational effects in dynamic proteins using a second-harmonic generation...

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• Biophysical screening identifies protein-ligand interactions but often fails to reveal functional consequences.
• Assessing functional changes, especially conformational shifts, can be complex and low-throughput.
• Acetyl choline binding protein (AChBP) serves as a model for ligand-gated ion channels (LGICs).

Purpose of the Study:

• To develop and validate a high-throughput second-harmonic generation (SHG) biosensor assay.
• To identify novel fragments that induce conformational changes in AChBP.
• To characterize the binding sites and kinetics of identified modulators.

Main Methods:

• Development of multiwell plate SHG assays using wild-type and mutant AChBP labeled with SHG-active tags.
• Screening of a 1056-fragment library against AChBP variants.
• Orthogonal validation using time-resolved grating-coupled interferometry and X-ray crystallography.

Main Results:

• SHG assays successfully detected distinct conformational changes induced by AChBP modulators.
• Fragment library screening yielded hit rates of 9-22%, identifying novel conformational modulators.
• Kinetic analysis revealed reversible 1:1 interactions, and crystallography confirmed binding to dynamic regulatory sites.

Conclusions:

• SHG biosensing is a sensitive, high-throughput method for detecting protein conformational changes.
• Novel fragments that modulate AChBP conformation and potentially LGIC function were identified.
• The study validates SHG as a powerful tool for fragment-based drug discovery targeting dynamic protein regions.