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Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules
Published on: November 2, 2009
Comparison of free-solution and surface-immobilized molecular interactions using a single platform
Ian R Olmsted1, Amanda Kussrow, Darryl J Bornhop
1Department of Chemistry and the Vanderbilt Institute for Chemical Biology, Vanderbilt University, 4226 Stevenson Center, Nashville, Tennessee 37235, USA.
Analytical Chemistry
|November 24, 2012
Summary
Surface immobilization impacts protein binding affinity. Backscattering interferometry (BSI) quantified how tethering concanavalin A (ConA) to a surface altered its binding to sugars, revealing closer proximity enhances affinity.
Area of Science:
- Biophysics
- Biochemistry
- Surface Science
Background:
- Surface immobilization is known to alter protein binding properties, but quantifying these effects has been challenging.
- Measuring binding affinities of molecules with significantly different masses, especially when immobilizing the larger molecule, presents technical hurdles.
Purpose of the Study:
- To quantify the effects of surface immobilization on protein binding affinity using a single platform.
- To investigate the relationship between the distance of protein tethering and binding affinity.
Main Methods:
- Utilized backscattering interferometry (BSI) to measure binding affinity of mannose and glucose to concanavalin A (ConA) in free solution.
- Chemically immobilized ConA on a sensor surface at varying distances (0, 7.17, and 20.35 nm) and measured binding constants using BSI.
Main Results:
- Concanavalin A (ConA) binding affinity to mannose and glucose was successfully quantified in free solution using picomoles of protein.
- Apparent binding affinity of the sugar-lectin pair increased as the distance between ConA and the sensor surface decreased.
Conclusions:
- Protein immobilization significantly affects binding constants, with closer proximity to the surface enhancing apparent affinity.
- These findings provide insights into variations in reported affinity values across different measurement techniques and immobilization strategies.
