Couette flow fluorescence detected linear dichroism for analytes in lipid bilayers

Abstract

Membranes are important sites of intermolecular interactions in biological systems. However, they present significant analytical challenges as they contain multiple analytes and are dynamic in nature. In this work, we show how a Jasco J‐1500 circular dichroism spectropolarimeter can be used with a microvolume Couette flow cell and appropriate cut‐off filters to measure excitation fluorescence detected linear dichroism (FDLD) of fluorophores embedded in liposomal membranes. The result is a spectrum that selectively probes the fluorophore(s) and eliminates the scattering that is apparent in the corresponding flow linear dichroism (LD) spectrum. The FDLD spectrum is opposite in sign from the LD spectrum with relative magnitudes modified by the quantum yields of the transitions. FDLD thus enables analyte orientations to be identified in a membrane. Data for a membrane peptide, gramicidin, and two aromatic analytes, anthracene and pyrene, are presented. Issues with the “leakage” of photons by the long pass filters used is also discussed.

Linear dichroism and linearly polarised luminescence spectra of oriented samples collected on a new integrated instrument

“…22 The positive short axis LD (378 nm) and negative long axis LD (254 nm) are in accord with the anthracenes aligning parallel to lipids thus perpendicular to the flow-direction. Intriguingly, as we previously observed, 14,26,27 anthracene in liposomes has much less coupling between the 254 nm and 378 nm transitions than in films (Fig. 1a) resulting in all LD components of the 378 nm band being positive.…”

“…The wavelength dependence of the dissymmetry factors for CD and CPL (ratios of differential and isotropic signals) was shown to provide more insight into the optical properties of the chiral lumiphores than either technique alone. In this work we present (as far as we are aware) the first wavelength scanning LD/LPL measurements, thus extending the advantages of LD [5][6][7][8][9][10][11][12][13][14][15][16][17] for high aspect ratio samples to luminescence. We show data produced for a fluorophore (anthracene) oriented on a stretched film and by binding to flow-oriented liposomes; and two fluorophores (the intercalator picogreen and groove binder 4 0 ,6-diamidino-2-phenylindole) oriented by binding to floworiented DNA.…”

Advantages of a synchrotron light source for fluorescence‐detected linear dichroism

“…We previously measured LD and FD-LD of anthracene on films and in liposomes. 1,3,10 Gudipati et al 19 with reference to prior research 20,21 provide a good summary of anthracene spectroscopy some of which forms the basis for the literature assignments of Table 1. In their own work, they used the polarized light of a synchrotron source and matrix isolation for the sample (15 K argon).…”

“…is often used to present data as it is independent of concentration and extinction coefficient. The FD-LD when collected with the voltage on the photomultiplier tube (PMT) fixed at a constant value and a long-pass cut-off filter inserted after the sample is directly proportional to the LD [1][2][3] FD À LD fixed HT,filter…”

“…For further details and also data collection in fixed-DC mode; see earlier studies. [1][2][3] This work was motivated by the fact that we are attracted to the ability of FD-LD to selectively probe any fluorophore(s) in an oriented sample, 1-3 but as with LD and circular dichroism (CD) on a bench-top instrument, our experiments suffered from loss of Xe-lamp light intensity below 200 nm. This work shows that synchrotron radiation (SR) does indeed deliver an extended wavelength range for measurement, as well as further advantages outlined below.…”