Mr. Christian Alejandro Iriarte Valdez Profile

Christian Alejandro Iriarte Valdez

at Leibniz Universität Hannover/ NIFE

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Proceedings Article | 1 April 2020 Presentation

Force spectroscopy measurements of ligand binding with transmembrane proteins using optical tweezers (Conference Presentation)

Carolin Riesenberg, Christian Alejandro Iriarte Valdez, Annegret Becker, Alexander Heisterkamp, Anaclet Ngezahayo, Maria Leilani Torres-Mapa

Proceedings Volume 11359, 1135915 (2020) https://doi.org/10.1117/12.2555811

KEYWORDS: Optical tweezers, Proteins, Spectroscopy, Particles, Receptors, Calibration, Feedback control, Silica, Laser optics, Thermodynamics

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Optical tweezers are powerful scientific tools capable of trapping and manipulating microscopic particles with a tightly focussed laser beam. They can be used to measure nm-sized movements as well as pN-forces in various applications . One of these applications is "dynamic force spectroscopy", where increasing forces are applied to e.g. receptor-ligand bonds to facilitate unbinding. Analysis of unbinding forces, or "rupture forces", can provide an overview of the thermodynamics and activation barriers during bond dissociation. Claudins are considered important component of tight junctions, which form a seal between epithelial cells to regulate paracellular transport. Clostridium perfringens enterotoxin (CPE) is a toxin that binds to certain claudins and disrupts the tight junctions. Understanding of the specific interaction between CPE and receptor claudins could lead to several pharmaceutical applications. This work aims to measure the binding strength between c-CPE and claudins in living cells, using an optical tweezers system. The system was characterized and calibrated extensively, which is necessary for force measurements. The trap stiffness in both lateral directions was measured for 1μm silica particles in cell culture medium. Stiffness values up to kx ≈ 89.81 pN/μm and ky ≈ 129.28 pN/μm in cell culture medium were determined. To investigate the interactions between C-CPE and claudins, 1 μm silica particles were coated with C-CPE and trapped with the optical tweezers. MCF-7 cells, which are known to express receptor claudins were positioned towards the particle until contact, then retracted. The technique can detect unbinding events and measure corresponding rupture forces from 1 pN up to 56.7 pN in magnitude. Overall, this work shows that optical tweezers offer a versatile and precise non-contact method to probe the interaction of transmembrane proteins in living cells.

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