Development of next-generation fluorescent probes is a key element in the quest for a greater understanding of complex biological environments (e.g., membranes) by bioimaging. Such fluorescence-based techniques rely on specialized small molecules that possess excellent fluorescent properties but also do not perturb the native biological environment in which they reside. Herein we present a theoretical/computational strategy for the design of novel optical probes for sensing in membranes based on the parent chromophore Nile Red. Using a combination of time-dependent density functional theory (TD-DFT) and molecular dynamics (MD), we have studied the optical properties and accommodation in a model membrane of Nile Red and eight analogs. Special attention has been given to the design of probes with improved solvatochromism and two-photon absorption (2PA) without altering the membrane properties. Of the eight studied analogs, two probes were found to possess attractive probe features and are hence suggested to be taken forward to chemical synthesis and experimental exploration.
Fluorescent probes are powerful tools for improving our understanding of cellular membranes and other complex biological environments. Using simulations, we gain atomistic and electronic insights into the effectiveness of the probes. In the current work, we have used various computational approaches to comprehensively investigate the properties of the fluorescent probe Laurdan and two Laurdan-like probes: AADAL and ECL. In addition, we propose the development of their corresponding novel malononitrile variants, which are computationally characterized herein. For the candidate probes, electronic structure calculations were used to rationalize their optical properties, including their ability for two-photon activation, and molecular dynamics simulations were used to unravel atomistic details of their functioning within lipid bilayers. While Laurdan, AADAL, and ECL were found to have very similar optical and membrane partitioning profiles, their malononitrile variants were found to show significantly improved optical properties, especially in regard to two-photon cross sections, and they appear to retain the desired membrane characteristics of the parent Laurdan molecule.
Accounting for solvent effects in theoretical predictions of spectroscopic properties may be of significant importance since a solvent -and on a more general basis any environment -may influence key spectroscopic parameters in nontrivial ways -especially in relation to calculation of non-linear optical properties. At the simplest level, solvent effects may be included into quantum chemistry calculations by use of a dielectric continuum approach, however, such a description may fail in addressing correctly environment anisotropies as well as specific interactions such as e.g. hydrogen bonding. On the other hand, discrete embedding methods allow for a more correct description of an environment since such methods keep the atomistic nature of the environment intact. In this paper, two-photon circular dichroism (TPCD) spectra will be calculated for two different biaryl derivatives introducing solvent effects by two different discrete embedding schemes, i.e. polarizable embedding based on induced dipoles (PE) or the fluctuating charge (FQ) model. While we find inclusion of solvent effects on this molecular property to be important, we conclude at the same time that the influence of the solvent on the TPCD rotatory strength is accounted for in an overall rather equivalent manner by either embedding methods.
Niemann Pick C2 (NPC2) is a small glycoprotein involved in cellular trafficking of cholesterol. Its dysfunction causes accumulation of cholesterol in the lysosomal organelles, a hallmark of Niemann Pick type C disease. Therefore, understanding cholesterol transport and the accumulation mechanism that may result is essential to understand the occurrence of this neurodegenerative disease. Cholestatrienol (CTL) is a fluorescent sterol widely used as an alternative to cholesterol because of its improved optical properties in spectroscopy and live-cell imaging. In this paper, the electronic circular dichroism (ECD) and the two-photon circular dichroism (TPCD) of CTL within the NPC2 binding pockets are simulated using response theory employing timedependent density functional theory (TD-DFT). Moreover, an explicit embedding method has been used to include the effect of the protein residues and the water solvent in calculating the spectroscopic properties. In both cases, an alteration of the CD signal is observed.
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