Spectral Properties and Orientation of Voltage-Sensitive Dyes in Lipid Membranes

Abstract

Voltage-sensitive dyes are frequently used for probing variations in the electric potential across cell membranes. The dyes respond by changing their spectral properties: measured as shifts of wavelength of absorption or emission maxima or as changes of absorption or fluorescence intensity. Although such probes have been studied and used for decades, the mechanism behind their voltage sensitivity is still obscure. We ask whether the voltage response is due to electrochromism as a result of direct field interaction on the chromophore or to solvatochromism, which is the focus of this study, as result of changed environment or molecular alignment in the membrane. The spectral properties of three styryl dyes, di-4-ANEPPS, di-8-ANEPPS, and RH421, were investigated in solvents of varying polarity and in model membranes using spectroscopy. Using quantum mechanical calculations, the spectral dependence of monomer and dimer ANEPPS on solvent properties was modeled. Also, the kinetics of binding to lipid membranes and the binding geometry of the probe molecules were found relevant to address. The spectral properties of all three probes were found to be highly sensitive to the local environment, and the probes are oriented nearly parallel with the membrane normal. Slow binding kinetics and scattering in absorption spectra indicate, especially for di-8-ANEPPS, involvement of aggregation. On the basis of the experimental spectra and time-dependent density functional theory calculations, we find that aggregate formation may contribute to the blue-shifts seen for the dyes in decanol and when bound to membrane models. In conclusion, solvatochromic and other intermolecular interactions effects also need to be included when considering electrochromic response voltage-sensitive dyes.

δ‐Peptides from RuAAC‐Derived 1,5‐Disubstituted Triazole Units

“…This peptide is long enough to stabilize H20 , resulting in the H14 , H16 and H20 helices being within 5 kcal/mol of each other in relative energy. To address the potential solubility of the different conformers, besides the polar DMSO used in the experimental investigations, the energies of the conformers were also obtained in the very polar aqueous matrix (Aq) and in the hydrophobic decanol, which can be used to approximate a lipid bilayer environment 28. We found that the three different solvents do not have a dramatic effect on the relative energy distribution of the conformers, and only some destabilization of H8 and H10 was observed when changing from the use of water to decanol as solvent.…”

Dyes as guests in ordered systems: current understanding and future directions

“…[24,25] As well as utilising dyes to probe the nature of the orientational order in membranes, guest dyes may be used, for example, to probe properties such as electric potentials across cell membranes, which relies on a detailed understanding of the alignment of the dye molecules within these ordered systems. [26,27] Despite being applied in very different contexts to the applications of guest-host liquid crystal systems described above, many of the underlying principles of molecular alignment and optical anisotropy within these systems are common to both areas of research. It is therefore important that the properties of anisotropic guest-host systems are well understood, but despite the attention they have received, there still remains a need to develop widely applicable quantitative structure-property relationships for these systems.…”

The orientation of a membrane probe from structural analysis by enhanced Raman scattering

“…28 Matson et al measured the orientation of di-4-ANEPPS and di-8-ANEPPS from linear dichroism measurements on flow-aligned vesicles using retinoic acid as a reference and found smaller polar angles of 14° and 18° for each, respectively. 29 Finally, Reeve et al used one photon, two photon, and second harmonic generation imaging on GUV's to measure both the polar tilt and its distribution. They found a polar angle of 52° for di-4-ANEPPS.…”

“…13 Note that the much smaller angle (14°) from linear dichroism is calibrated to measurements on retinol, which is presumed to be perfectly normal based on measurements on the retinol group in bacteriorhodopsin. 29 It is possible that free retinol in a membrane is at a tilted angle (like all other optical probes studied to date), which would increase the estimated angles of di-4-ANEPPS and di-8-ANEPPS by that method. A unique aspect of SABERS is that it finds the molecular roll angle in addition to the polar tilt within the bilayer since it is based on the alignment of the full polarizability tensors rather than just estimates of a property along the molecule.…”

“…[21][22][23][24][25] The molecular orientation of di-8-ANEPPS and di-4-ANEPPS have been studied with polarized fluorescence and second harmonic generation (SHG), as well as linear dichroism on aligned lipid membranes. 13,[26][27][28][29] These measurements give a range of conflicting answer for the probe tilt from the membrane normal. Here we apply a method we recently introduced called structural analysis by enhanced Raman scattering (SABERS) to study the probe orientation.…”