Influence of Membrane Phase on the Optical Properties of DPH

The fluorescent molecule diphenylhexatriene (DPH) has been often used in combination with fluorescence anisotropy measurements, yet little is known regarding the non-linear optical properties. In the current work, we focus on them and extend the application to fluorescence, while paying attention to the conformational versatility of DPH when it is embedded in different membrane phases. Extensive hybrid quantum mechanics/molecular mechanics calculations were performed to investigate the influence of the phase- and temperature-dependent lipid environment on the probe. Already, the transition dipole moments and one-photon absorption spectra obtained in the liquid ordered mixture of sphingomyelin (SM)-cholesterol (Chol) (2:1) differ largely from the ones calculated in the liquid disordered DOPC and solid gel DPPC membranes. Throughout the work, the molecular conformation in SM:Chol is found to differ from the other environments. The two-photon absorption spectra and the ones obtained by hyper-Rayleigh scattering depend strongly on the environment. Finally, a stringent comparison of the fluorescence anisotropy decay and the fluorescence lifetime confirm the use of DPH to gain information upon the surrounding lipids and lipid phases. DPH might thus open the possibility to detect and analyze different biological environments based on its absorption and emission properties.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

PIP2 Influences the Conformational Dynamics of Membrane bound KRAS4b

ABSTRACTKRAS4b is a small GTPase involved in cellular signaling through receptor tyrosine kinases. Activation of KRAS4b is achieved through the interaction with nucleotide exchange factors while inactivation is regulated by through interaction with GTPase activating proteins. The activation of KRAS4b only occurs after recruitment of the regulatory proteins to the plasma membrane thus making the role of the phospholipid bilayer an integral part of the activation mechanism. The phospholipids, primarily with anionic head groups, interact with both the membrane anchoring hypervariable region and the G-domain, thus influencing the orientation of KRAS at the membrane surface. The orientation of the G-domain at the membrane surface is believed to play a role in the regulation of KRAS activation. Much of the research has focused on the role of phosphatidyl serine but little has been done regarding the important signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2). We report here the use of fluorescence anisotropy decay, atomic force microscopy, and molecular dynamic simulations to show that the presence of PIP2 in the bilayer promotes the interaction of the G-domain with the bilayer surface. The stability of these interactions significantly alters the dynamics of KRAS4b bound to the membrane indicating a potential role for PIP2 in the regulation of KRAS4b activity.

Comment on ““Where does the fluorescing moiety reside in a carbon dot?” – Investigations based on fluorescence anisotropy decay and resonance energy transfer dynamics” by A. Das, D. Roy, C. K. De and P. K. Mandal, Phys. Chem. Chem. Phys., 2018, 20, 2251

In a recent paper published in Physical Chemistry Chemical Physics (Phys. Chem. Chem. Phys., 2018, 20, 2251–2259), Förster resonance energy transfer (FRET) between carbon dots and rhodamine 123 has been reported.

Orientational distribution of DPH in lipid membranes: a comparison of molecular dynamics calculations and experimental time-resolved anisotropy experiments

The behavior of the fluorescent probe diphenylhexatriene (DPH) in different lipid phases is investigated. The rotational autocorrelation functions are calculated in order to model the time-resolved fluorescence anisotropy decay. The role of the order parameters is discussed.

Reply to the ‘Comment on ““Where does the fluorescing moiety reside in a carbon dot?” – Investigations based on fluorescence anisotropy decay and resonance energy transfer dynamics”’ by H. C. Joshi, Phys. Chem. Chem. Phys., 2019, 21, DOI: 10.1039/c9cp00136k

Claims made in the Comment are perhaps incorrect and misleading. These claims have been negated with proper analytical reasoning.

“Where does the fluorescing moiety reside in a carbon dot?” – Investigations based on fluorescence anisotropy decay and resonance energy transfer dynamics

It has been shown recently that aggregated dyes are responsible for very high fluorescence in a carbon dot (CD). Location of the fluorescing unit in a carbon dot could be shown.