Abstract:This paper describes a multivariate photokinetic analysis of the membrane phase dependence of PRODAN and LAURDAN photokinetics in DMPC vesicles. Decay data, arranged in the form of Fourier transformed emission-decay matrices (FT-EDMs), were collected as a function of temperature around the gel phase transition temperature. Each matrix was partitioned into the emission spectra and decay profiles of the underlying emission components using methods based on principal components analysis. The analysis revealed tha… Show more
“…In addition, water molecules may also form hydrogen bonds to BADAN, and the BADAN moiety itself may undergo a conformational change from a relatively planar to a relatively twisted excited state. Both hydrogen bonding (Artukhov et al 2007; Balter et al 1988; Chapman et al 1995; Józefowicz et al 2005; Parasassi and Gratton 1995; Rowe and Neal 2006; Samanta and Fessenden 2000; Viard et al 1997) and conformational twisting (Abbyad et al 2007; Lobo and Abelt 2003; Parusel et al 1998; Vincent et al 2005) were found to contribute to the relaxation of the fluorescence of the well-known lipid probes PRODAN and LAURDAN, whose chromophore moieties are identical to that of BADAN.Fig. 2Experimental ( a , b ) and reconstructed streak camera images ( c , d ) of BADAN-labeled M13 coat protein mutants having the label in the polar (G03C, a and c ) and hydrophobic core (V29C, b and d ) region of mixed PC/PG phospholipid bilayers.…”
Profiles of lipid-water bilayer dynamics were determined from picosecond time-resolved fluorescence spectra of membrane-embedded BADAN-labeled M13 coat protein. For this purpose, the protein was labeled at seven key positions. This places the label at well-defined locations from the water phase to the center of the hydrophobic acyl chain region of a phospholipid model membrane, providing us with a nanoscale ruler to map membranes. Analysis of the time-resolved fluorescence spectroscopic data provides the characteristic time constant for the twisting motion of the BADAN label, which is sensitive to the local flexibility of the protein–lipid environment. In addition, we obtain information about the mobility of water molecules at the membrane–water interface. The results provide an unprecedented nanoscale profiling of the dynamics and distribution of water in membrane systems. This information gives clear evidence that the actual barrier of membranes for ions and aqueous solvents is located at the region of carbonyl groups of the acyl chains.
“…In addition, water molecules may also form hydrogen bonds to BADAN, and the BADAN moiety itself may undergo a conformational change from a relatively planar to a relatively twisted excited state. Both hydrogen bonding (Artukhov et al 2007; Balter et al 1988; Chapman et al 1995; Józefowicz et al 2005; Parasassi and Gratton 1995; Rowe and Neal 2006; Samanta and Fessenden 2000; Viard et al 1997) and conformational twisting (Abbyad et al 2007; Lobo and Abelt 2003; Parusel et al 1998; Vincent et al 2005) were found to contribute to the relaxation of the fluorescence of the well-known lipid probes PRODAN and LAURDAN, whose chromophore moieties are identical to that of BADAN.Fig. 2Experimental ( a , b ) and reconstructed streak camera images ( c , d ) of BADAN-labeled M13 coat protein mutants having the label in the polar (G03C, a and c ) and hydrophobic core (V29C, b and d ) region of mixed PC/PG phospholipid bilayers.…”
Profiles of lipid-water bilayer dynamics were determined from picosecond time-resolved fluorescence spectra of membrane-embedded BADAN-labeled M13 coat protein. For this purpose, the protein was labeled at seven key positions. This places the label at well-defined locations from the water phase to the center of the hydrophobic acyl chain region of a phospholipid model membrane, providing us with a nanoscale ruler to map membranes. Analysis of the time-resolved fluorescence spectroscopic data provides the characteristic time constant for the twisting motion of the BADAN label, which is sensitive to the local flexibility of the protein–lipid environment. In addition, we obtain information about the mobility of water molecules at the membrane–water interface. The results provide an unprecedented nanoscale profiling of the dynamics and distribution of water in membrane systems. This information gives clear evidence that the actual barrier of membranes for ions and aqueous solvents is located at the region of carbonyl groups of the acyl chains.
“…This observation requires the ultrafast methods employed in the present work, demonstrating the complementarity of the approach to earlier measurements using other types of time-dependent spectroscopies. 6,26,27 The sensitivity of the relaxation dynamics to the local molecular environment suggests that the approach will yield new information on the local environment of polarity sensitive dyes when applied to biomembranes.…”
Lipiophilic dyes such as Laurdan and Prodan are widely used in membrane biology due to a strong bathochromic shift in emission that reports structural parameters of the membrane such as area per molecule. Disentangling the factors which control the spectral shift is complicated by the stabilization of a charge-transfer-like excitation of the dye in polar environments. Predicting the emission therefore requires modeling both the relaxation of the environment and the corresponding evolution of the excited state. Here an approach is presented in which (i) the local environment is sampled by classical molecular dynamics (MD) simulation of the dye and solvent; (ii) the electronically excited state of Prodan upon light absorption is predicted by numerical quantum mechanics (QM); (iii) iterative relaxation of the environment around the excited dye by MD coupled with evolution of the excited state is performed; (iv) the emission properties are predicted by QM. The QM steps are computed using many-body Green's functions theory in the GW approximation and the Bethe-Salpeter Equation with the environment modeled as fixed point charges, sampled in the MD simulation steps. Comparison to ultrafast time resolved transient absorption measurements demonstrates that the iterative MM/QM approach agrees quantitatively with both the polarity dependent shift in emission and the timescale over which the charge transfer state is stabilized. Together the simulations and experimental measurements suggest that evolution into the charge-transfer state is slower in amphiphilic solvents. File list (2) download file view on ChemRxiv maintext.pdf (3.25 MiB) download file view on ChemRxiv SI.pdf (7.99 MiB) Ultrafast formation of the charge-transfer state of prodan reveals unique aspects of the chromophore environment
“…Recently, solvent relaxation studies were carried out in microviscous media such as vesicles or sol−gel glasses. , The authors have shown that 6-propionyl-2-(dimethylamino)naphthalene (prodan) is a suitable probe for this type of research. In this study, we used two probes, (i) prodan and (ii) laurdan , (Chart ), that have the same fluorescent portion but differ substantially in affinity to the studied nanoparticles due to the absence/presence of a long aliphatic tail. The aim of the study was two-fold: (i) we compare results obtained for both probes and analyze to what extent the partitioning of prodan between vesicles and bulk solvent affects the relaxation and complicates the data analysis and (ii) we present a piece of evidence that the lateral motion of the probe with respect to the nanoparticle within its lifetime is a significant part of the slow relaxation processes in systems containing polymeric nanoparticles.…”
Steady-state and time-resolved fluorescence measurements were used to study the relaxation of the microenvironment of hydrophobic probes 6-propionyl-2-(dimethylamino)naphthalene (prodan) and 6-dodecanoyl-2-(dimethylamino)naphthalene (laurdan) in systems containing vesicles formed by the amphiphilic diblock copolymer poly(epsilon-caprolactone)-block-poly(ethylene oxide) (PCL-PEO) and water/tetrahydrofurane (THF) solvent mixtures. It was found that in case of prodan, both steady-state and time-resolved emission spectra were composed of two subspectra corresponding to the emission of prodan molecules located (i) in fairly rigid (effectively viscous) and hydrophobic domains of the vesicles close to the PCL/PEO interface and (ii) in a more polar and less viscous medium (in the bulk solution). The fraction of the emission from the more polar microenvironment increases with increasing content of THF in the system. Laurdan, in contrast to prodan, appeared to be solubilized preferentially in the hydrophobic domains up to 30 vol % of THF content, and its emission spectra changed only due to swelling of hydrophobic PCL domains by added THF. The study shows that the analysis of the time-resolved emission from a probe distributed in two media is, in principle, possible, but it is quite complex and appreciably less accurate, and the relaxation times are ill-defined averages of several processes. The bimodal or shoulder-containing time-resolved spectra have to be decomposed in pertinent time-resolved subspectra and treated separately. Another important result of the study is a piece of knowledge concerning the motion of the probe with respect to the vesicle. In the studied complex system, not only the relaxation of the solvent and reorganization of polymer segments around the fluorescent headgroup of the probe affect the emission but also a lateral motion of the probe with respect to the nanoparticle within the lifetime of the excited state contributes significantly to the relaxation and to the relatively slow time-resolved Stokes shift.
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