Showing papers by "Daniel Wüstner published in 2020"

One- and two-photon solvatochromism of the fluorescent dye Nile Red and its CF3, F and Br-substituted analogues

Abstract: The solvatochromic fluorophore Nile Red, 9-diethylamino-5H-benzo[a]phenoxazine-5-one, is one of the most commonly used stains to enhance contrast of lipid-rich areas of microscopic biosamples. Quite surprisingly, relatively little is known about the spectrally-resolved two-photon absorption (2PA) properties of this dye despite its promising features for two-photon microscopy of biological matter. For this reason, the two-photon solvatochromism of Nile Red still remains an uncharted territory as well. Also, no study has yet reported on how electron-withdrawing substituents attached to the Nile Red backbone affect its solvatochromic properties and two-photon brightness. In this paper, we demonstrate how solvent polarity influences the one- and two-photon absorption spectra of Nile Red as well as its fluorescence parameters, and we present new analogues that contain –CF3, –F and –Br substituents on its eastern side. Two-photon excited fluorescence experiments in a broad spectral range (780–1240 nm) and electronic structure calculations show that both the nature and location of the substituent have particular influence on the strength of 2PA, peaking in all cases at approx. 860 and 1050 nm. 2PA cross sections are higher at 1050 nm than at 860 nm, which suggests that Nile Red and its analogues are best suited for two-photon imaging employing excitation in the NIR-II optical transparency window of biological tissues.

Cholesterol binding to the sterol-sensing region of Niemann Pick C1 protein confines dynamics of its N-terminal domain

Abstract: Lysosomal accumulation of cholesterol is a hallmark of Niemann Pick type C (NPC) disease caused by mutations primarily in the lysosomal membrane protein NPC1. NPC1 contains a transmembrane sterol-sensing domain (SSD), which is supposed to regulate protein activity upon cholesterol binding, but the mechanisms underlying this process are poorly understood. Using atomistic simulations, we show that in the absence of cholesterol in the SSD, the luminal domains of NPC1 are highly dynamic, resulting in the disengagement of the NTD from the rest of the protein. The disengaged NPC1 adopts a flexed conformation that approaches the lipid bilayer, and could represent a conformational state primed to receive a sterol molecule from the soluble lysosomal cholesterol carrier NPC2. The binding of cholesterol to the SSD of NPC1 allosterically suppresses the conformational dynamics of the luminal domains resulting in an upright NTD conformation. The presence of an additional 20% cholesterol in the membrane has negligible impact on this process. The additional presence of an NTD-bound cholesterol suppresses the flexing of the NTD. We propose that cholesterol acts as an allosteric effector, and the modulation of NTD dynamics by the SSD-bound cholesterol constitutes an allosteric feedback mechanism in NPC1 that controls cholesterol abundance in the lysosomal membrane.

Mechanistic Insight into Lipid Binding to Yeast Niemann Pick Type C2 Protein.

Abstract: Niemann Pick type C2 (NPC2) is a small sterol binding protein in the lumen of late endosomes and lysosomes. We showed recently that the yeast homologue of NPC2 together with its binding partner NCR1 mediates integration of ergosterol, the main sterol in yeast, into the vacuolar membrane. Here, we study the binding specificity and the molecular details of lipid binding to yeast NPC2. We find that NPC2 binds fluorescence- and spin-labeled analogues of phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), and sphingomyelin. Spectroscopic experiments show that NPC2 binds lipid monomers in solution but can also interact with lipid analogues in membranes. We further identify ergosterol, PC, and PI as endogenous NPC2 ligands. Using molecular dynamics simulations, we show that NPC2's binding pocket can adapt to the ligand shape and closes around bound ergosterol. Hydrophobic interactions stabilize the binding of ergosterol, but binding of phospholipids is additionally stabilized by electrostatic interactions at the mouth of the binding site. Our work identifies key residues that are important in stabilizing the binding of a phospholipid to yeast NPC2, thereby rationalizing future mutagenesis studies. Our results suggest that yeast NPC2 functions as a general "lipid solubilizer" and binds a variety of amphiphilic lipid ligands, possibly to prevent lipid micelle formation inside the vacuole.

Modeling the Sterol-Binding Domain of Aster-A Provides Insight into Its Multiligand Specificity.

Abstract: Intracellular transport of cholesterol and related sterols relies to a large degree on nonvesicular mechanisms, which are only partly understood at the molecular level. Aster proteins belonging to the Lam family of sterol transfer proteins have recently been identified as important catalysts of nonvesicular sterol exchange between the plasma membrane (PM) and endoplasmic reticulum (ER). Here, we used a range of computational tools to study the molecular mechanisms underlying sterol binding as well as multisterol ligand specificity of Aster-A. Our study focused primarily on gaining atomistic insight into the bound ligand-protein complex and was, on this basis, performed in the absence of any membrane. Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations provide a rationale for the experimentally found ranking of binding affinities of various sterols to Aster-A. In particular, the polarity of the sterols and the length of their alkyl chain could be identified as being critical determinants of ligand affinity. A Gibbs free energy decomposition identified a charged residue, Glu444, at the base of the binding pose as an important control point for sterol binding. Removing its net charge via protonation was found to cause significant changes to the environment surrounding this residue. In addition, the protonation of Glu444 was found to be paralleled by a large redistribution of molecular flexibility in the Aster domain. This finding was supplemented by multiple branched adaptive steered molecular dynamics (MB-ASMD) simulations by which we defined a possible molecular path for sterol release and demonstrated the importance of Glu444 in this process.