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Dual polarization interferometry characterization of carbohydrate-protein interactions
Sylvie Ricard-Blum1, Louise L Peel, Florence Ruggiero
1Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-UCBL, 7 passage du Vercors 69367, Lyon cedex 07, France.
Insights
Dual polarization interferometry (DPI) precisely measured carbohydrate-protein interactions, revealing surface loss instead of conformational changes during HepV-heparin binding. This advanced technique offers a clearer understanding of binding mechanisms.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biophysics
Background:
- Carbohydrate-protein interactions are crucial in biological processes.
- Heparin-protein interactions, specifically with collagen V (HepV), are biologically relevant but kinetically complex.
- Previous studies using Surface Plasmon Resonance (SPR) suggested conformational changes in HepV-heparin binding.
Purpose of the Study:
- To analyze the binding of HepV to heparin using Dual Polarization Interferometry (DPI).
- To determine the thickness, density, and mass of surface structures during HepV-heparin binding in real time.
- To investigate the anomalous kinetic behavior observed in SPR assays and elucidate the binding mechanism.
Main Methods:
- Utilized Dual Polarization Interferometry (DPI) for real-time analysis of molecular binding.
- Immobilized a streptavidin layer on a sensor surface, followed by capturing biotinylated heparin.
- Measured the binding of HepV to the immobilized heparin-streptavidin complex.
Main Results:
- DPI successfully quantified the binding of HepV to heparin, determining a stoichiometric ratio of approximately 1.7:1.0.
- Real-time DPI analysis indicated surface loss (likely streptavidin) during binding, rather than conformational changes.
- The technique allowed for reliable stoichiometric ratio calculation, which was challenging with SPR.
Conclusions:
- DPI provides a robust method for analyzing carbohydrate-protein interactions, including thickness, density, and mass.
- The HepV-heparin binding mechanism involves surface loss, offering a different perspective from previous SPR-based interpretations.
- This study highlights DPI's utility in resolving complex binding kinetics and elucidating molecular interactions.
Abstract:
Dual polarization interferometry (DPI) is an analytical technique that allows the simultaneous determination of thickness, density, and mass of a biological layer on a sensing waveguide surface in real time. The technique was applied to the analysis of carbohydrate-protein interactions. The selected system involved a 12-kDa recombinant fragment of collagen V (HepV) and heparin, a complex polysaccharide. Here we report on the analysis of thickness, density, and mass of surface structures obtained during the binding of HepV to heparin, which is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. This system, which was initially studied for its biological relevance, displayed anomalous behavior in kinetic studies using surface plasmon resonance (SPR) assays that has been attributed to putative conformational changes. It was this putative conformational change that prompted us to investigate the binding using an alternative analytical approach. While using DPI to monitor binding events, a streptavidin layer (surface coverage 2.105 ng mm(-2)) was bound to the sensor surface (92% coverage), which captured 0.105 ng mm(-2) of biotinylated heparin (a stoichiometric ratio of 1:6 heparin-streptavidin). The heparin inserted into the streptavidin layer but was still found to be capable of binding 0.154 ng mm(-2) of HepV, which was also observed to insert into the streptavidin layer. This allowed the reliable calculation of the stoichiometric ratio for the HepV-heparin complex ( approximately 1.7:1.0), which has proved to be difficult to evaluate by SPR assays. Furthermore, real-time analysis of the heparin-HepV interaction by DPI suggested that there was some surface loss (probably of streptavidin) while the binding was occurring rather than the putative conformational change that has been suggested on the basis of kinetic data alone. This gives further insight into the binding mechanism of HepV to heparin.
