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Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)
Published on: November 29, 2014
Using receptor conformational change to detect low molecular weight analytes by surface plasmon resonance
J E Gestwicki1, H V Hsieh, J B Pitner
1BD Technologies, Research Triangle Park, North Carolina 27709, USA.
Analytical Chemistry
|January 5, 2002
Summary
This study demonstrates a new surface plasmon resonance (SPR) method to detect small molecules by monitoring protein conformational changes, enabling direct detection of low molecular weight analytes like calcium ions.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biophysics
Background:
- Small molecules are challenging to detect with standard surface plasmon resonance (SPR) due to their low molecular weight, which yields insufficient refractive index changes.
- While SPR can detect protein conformational changes, this has not been utilized for small molecule detection.
- Existing methods often require modifications to standard SPR instruments for small molecule analysis.
Purpose of the Study:
- To develop a novel SPR-based method for the direct detection of small molecules by leveraging ligand-induced conformational changes in immobilized proteins.
- To demonstrate the applicability of this technique for monitoring the binding of low molecular weight ligands to protein receptors.
- To validate the method's effectiveness and potential for high-throughput screening and biosensor development.
Main Methods:
- Immobilized maltose-binding protein and tissue transglutaminase were used as receptors on SPR sensors.
- Ligand binding was monitored by observing changes in refractive index resulting from altered hydrodynamic radius of the receptor proteins.
- SPR responses were analyzed for specificity, reversibility, and compared with known dissociation constants.
Main Results:
- Ligand binding induced measurable refractive index changes due to conformational shifts, not just mass addition.
- A decrease in hydrodynamic radius resulted in a refractive index decrease, while an increase caused a positive change.
- The SPR method successfully detected specific small molecule ligands, including calcium ions (40 Da), with high accuracy.
- SPR-determined equilibrium dissociation constants correlated well with reported values.
Conclusions:
- Ligand-induced conformational changes provide a viable mechanism for direct SPR detection of small molecules without instrument modification.
- This technique overcomes limitations of traditional SPR for low molecular weight analyte detection.
- The method holds significant potential for applications in drug discovery, high-throughput screening, and the development of novel biosensors.
