Structural design of intrinsically fluorescent oxysterols

Abstract

Oxysterols are oxidized derivatives of cholesterol with many important biological functions. Trafficking of oxysterols in and between cells is not well studied, largely due to the lack of appropriate oxysterol analogs. Intrinsically fluorescent oxysterols present a new route towards direct observation of intracellular oxysterol trafficking by fluorescence microscopy. We characterize the fluorescence properties of the existing fluorescent 25-hydroxycholesterol analog 25-hydroxycholestatrienol, and propose a new probe with an extended conjugated system. The location of both probes inside a membrane is analyzed and compared with that of 25-hydroxycholesterol using molecular dynamics simulations. The analogs' one- and two-photon absorption properties inside the membrane are evaluated using electronic structure calculations with polarizable embedding. Due to predicted keto-enol tautomerisation of the new oxysterol analog, we also evaluate the keto form. Both analogs are found to be good probe candidates for 25-hydroxycholesterol, provided that the new analog remains in the enol-form. Only the new analog with extended conjugated system shows significant two-photon absorption, which is strongly enhanced by the presence of the membrane.

Modeling the Sterol-Binding Domain of Aster-A Provides Insight into Its Multiligand Specificity

“…Furthermore, we have analyzed the binding of the intrinsically fluorescent sterols DHE, cholestatrienol (CTL) and 25-hydroxy-CTL. These sterols differ from ergosterol, cholesterol, and 25-OH-cholesterol, respectively, only by having additional double bonds in the sterol ring system giving them slight fluorescence for spectroscopic and imaging studies of protein–sterol interactions. − Moreover, Aster-A has recently been shown to transfer DHE efficiently between membranes . We included fluorescent 22-NBD-cholesterol since previous sterol binding and competition assays used it as a reference ligand.…”

The Secret Lives of Fluorescent Membrane Probes as Revealed by Molecular Dynamics Simulations

“…Previous to this latter study, the same research group had also used the same computational protocols to study 25-hydroxycholesterol ( Figure 14 a) and its previously synthesized fluorescent analogue 25-hydroxycholestatrienol (25-OH-CTL; Figure 14 b), as well as the novel compound equivalent to C4P, with a 25-hydroxyl group (25-OH-C4P) [ 156 ]. 25-OH-CTL was confirmed as a good fluorescent analogue of 25-hydroxycholesterol, in terms of location, orientation, and effects on membrane ordering and thickness.…”

Photophysical and Structural Characterization of Intrinsically Fluorescent Sterol Aggregates

“…It differs from cholesterol by having two additional double bonds in the ring system and by bearing an extra methyl group and a double bond in the alkyl side chain (Figure A). , Because of this close similarity to cholesterol, DHE has been employed in the past to study sterol aggregation in solution and model and cell membranes. − Given its extensive characterization in model and cell membranes, its similarity to cholesterol and ergosterol (the main sterols in eukaryotic cells), and its commercial availability, DHE can be considered a prototype of intrinsically fluorescent sterol probes. Using live-cell imaging of DHE, vesicular and nonvesicular sterol transport modes have been discovered in mammalian cells and yeast cells, which could be confirmed for cholesterol and ergosterol. − The related cholestatrienol (CTL), as well as the recently developed side-chain oxidized CTL derivatives, share the three-conjugated double-bond “chromophore” with DHE and therefore have almost identical optical properties. − However, those probes are not commercially available and therefore are less accessible than DHE, which is widely used in the biophysics and cell biology community, e.g., to characterize protein–sterol interactions . A systematic analysis of DHE’s photophysical properties in its monomeric and aggregated form is necessary to interpret such experiments, but only a few studies have addressed this problem so far.…”