Nonlinear Light Scattering as a Generally Applicable Approach for Studying Molecular Transport across Biological Membranes

Abstract

Characterization of molecular interactions with biological membranes is of fundamental biological importance. We have previously demonstrated use of second-harmonic light scattering (SHS), a phenomenon based upon second-harmonic generation (SHG)1–4, as a real-time measure of molecular uptake in living cells. A critical limitation of SHS for characterizing membrane-specific transport is that the molecule-of-interest must be SHG-active (i.e. possessing measurable optical nonlinear polarizability). This is particularly severe as many biologically significant molecules are SHG-inactive. To circumvent this limitation, we describe here a proof-of-concept study establishing competitive transport as a means of extending SHS to quantify SHG-inactive species. Briefly, the measured transport kinetics of an SHG-active molecule can be perturbed by the co-presence of an SHG-inactive species competing to cross a common barrier. Deconvolution of the perturbed signal yields the transport rate of the SHG-inactive species. As a representative example, we examine competitive transport of a strong SHG-active cation, malachite green (MG+), against the weakly SHG-active dication, propidium (Pro2+), across the outer-membrane protein channels in E. coli. Compared to MG+, Pro2+ produces a negligible SHS signal. However, as it is SHG-active, Pro2+ transport can be measured directly. A one-site channel model is developed to extract the Pro2+ transport rate from the perturbed MG+ signal, and is validated against the directly measured rate.REFERENCES1. Zeng, J., et al., Biophys. J. 104: 139–145 (2013).2. Wilhelm, M.J., et al., Chem. Phys. Lett. 605-606: 158–163 (2014).3. Wilhelm, M.J., et al., ACS Chem. Biol. 10: 1711−1717 (2015).4. Wilhelm, M.J., et al., Biochemistry 54: 4427-4430 (2015).