Laurdan Spectrum Decomposition as a Tool for the Analysis of Surface Bilayer Structure and Polarity: a Study with DMPG, Peptides and Cholesterol

Abstract

The highly hydrophobic fluorophore Laurdan (6-dodecanoyl-2-(dimethylaminonaphthalene)) has been widely used as a fluorescent probe to monitor lipid membranes. Actually, it monitors the structure and polarity of the bilayer surface, where its fluorescent moiety is supposed to reside. The present paper discusses the high sensitivity of Laurdan fluorescence through the decomposition of its emission spectrum into two Gaussian bands, which correspond to emissions from two different excited states, one more solvent relaxed than the other. It will be shown that the analysis of the area fraction of each band is more sensitive to bilayer structural changes than the largely used parameter called Generalized Polarization, possibly because the latter does not completely separate the fluorescence emission from the two different excited states of Laurdan. Moreover, it will be shown that this decomposition should be done with the spectrum as a function of energy, and not wavelength. Due to the presence of the two emission bands in Laurdan spectrum, fluorescence anisotropy should be measured around 480 nm, to be able to monitor the fluorescence emission from one excited state only, the solvent relaxed state. Laurdan will be used to monitor the complex structure of the anionic phospholipid DMPG (dimyristoyl phosphatidylglycerol) at different ionic strengths, and the alterations caused on gel and fluid membranes due to the interaction of cationic peptides and cholesterol. Analyzing both the emission spectrum decomposition and anisotropy it was possible to distinguish between effects on the packing and on the hydration of the lipid membrane surface. It could be clearly detected that a more potent analog of the melanotropic hormone alpha-MSH (Ac-Ser(1)-Tyr(2)-Ser(3)-Met(4)-Glu(5)-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2)) was more effective in rigidifying the bilayer surface of fluid membranes than the hormone, though the hormone significantly decreases the bilayer surface hydration.

Relationships between membrane water molecules and Patman equilibration kinetics at temperatures far above the phosphatidylcholine melting point

“…In each case, the GP value decreased continuously as a function of temperature above the main transition into the L α phase. These results are consistent with others reported previously for Laurdan [4,13,17,19], although this may be the first time that it has been shown that the phenomenon occurs even at temperatures more than 90°C above the melting temperature (open squares in Panel B, dipalmitoleoylphosphatidylcholine melting temperature = −36°C).…”

“…Studies of either saturated phosphatidylcholine or phosphatidylglycerol consistently show a steady increase of apparent membrane polarity (i.e. decreased GP value or stronger solvent relaxation) as a function of temperature well above the lipid melting point [4,13,19].…”

“…These strong solvent effects have been used to provide indirect estimations of membrane order and fluidity, which appear to correlate with water invasion into the bilayer [11,12]. Typically, these solvent effects are quantified by calculating a parameter termed "Generalized Polarization" (GP) [4,11] or by deconvolution of spectral components by non-linear regression [13,14]. With these methods, physical phenomena such as lipid phases have been readily observed [4,11,[15][16][17][18].…”

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

“…There is a wealth of studies on model membranes30313233343536 and an emerging literature12363738 on the use of dynamic imaging approaches to measure spectral relaxation in living cells. At the time this work was being completed, the Gratton laboratory12 published a phasor-based approach to measure membrane polarity and microviscosity.…”

Interactions of Lipid Membranes with Fibrillar Protein Aggregates

“…In a lipid bilayer, this amphiphilic fluorophore is localized at the level of the glycerol backbone, with lauric acid tail anchored in the acyl chain region. Laurdan's emission spectrum has two clearly distinct components that are attributed to solvent-unrelaxed (shorter-wavelength band centered circa 440 nm) and solvent-relaxed states (longer-wavelength band centered circa 490 nm) (Lúcio et al 2010;Sanchez et al 2012). This photophysical property is thought to originate from the reorientation of water dipoles around the excited-state dipole of the probe molecule.…”