Related Experiment Video
Updated: Jun 12, 2025
09:43
Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy
Published on: August 13, 2019
9.4K
Ligand-induced conformational changes in protein molecules detected by sum-frequency generation
Joshua Salafsky1, Patrik K Johansson2, Elwy Abdelkader3
1Department of Pharmaceutical Chemistry, University of California, San Francisco (UCSF), San Francisco, California; Skylight Discovery, Inc., Suite 300, Seattle, Washington.
Biophysical Journal
|September 21, 2024
Summary
Sum-frequency generation (SFG) spectroscopy detects ligand-induced protein conformational changes. This novel method maps molecular dynamics and allosteric networks, aiding drug discovery by identifying ligand binding sites.
Area of Science:
- Biophysics
- Spectroscopy
- Molecular Biology
Background:
- Understanding protein conformational changes is crucial for deciphering biological functions and mechanisms of drug action.
- Existing methods for studying these dynamics can be limited in sensitivity or perturb the biological system.
Purpose of the Study:
- To demonstrate sum-frequency generation (SFG) spectroscopy as a method for detecting ligand-induced conformational changes in proteins.
- To establish a technique for mapping allosteric networks and identifying ligand binding sites.
Main Methods:
- Utilized isotope-labeled amino acids and cell-free synthesis to create deuterated KRas(G12D) protein constructs.
- Immobilized proteins on supported bilayer membranes to ensure ordered molecular layers.
- Employed sum-frequency generation (SFG) spectroscopy to detect vibrational signals from labeled and unlabeled proteins before and after inhibitor binding.
Main Results:
- Achieved exceptionally large SFG amide I signals, indicating high orientational order of proteins on the bilayer.
- Observed distinct CDx signals in deuterated proteins, sensitive to inhibitor binding.
- Detected significant shifts in CDx SFG intensity for alanine, valine, and glycine residues upon peptide inhibitor (KRpep-2d) binding, correlating with known structural data.
Conclusions:
- Sum-frequency generation (SFG) spectroscopy can effectively detect ligand-induced conformational changes in proteins.
- The technique provides a sensitive, nonperturbative approach to map allosteric networks and identify ligand binding sites.
- This method holds promise for structure-function relationship studies and drug discovery.
