This study explores the structural and electronic factors affecting the absorption spectra of 5-carboxy-tetramethylrhodamine (TAMRA) in water, a widely used fluorophore in imaging and molecular labeling in biophysical studies. Through molecular dynamics (MD) simulations and density functional theory (DFT) calculations, we examine TAMRA UV absorption spectra together with TAMRA-labeled peptides (Arg9, Arg4, Lys9). We found that DFT calculations with different functionals underestimate TAMRA maximum UV absorption peak by ~100 nm, resulting in the maximum at ca. 450 nm instead of the experimental value of ca. 550 nm. However, incorporating MD simulation snapshots of TAMRA in water, the UV maximum peak shifts and is in close agreement with the experimental results due to the rotation of TAMRA N(CH3)2 groups, effectively captured in MD simulations. The method is used to estimate the UV absorption spectra of TAMRA-labeled peptides, matching experimental values.

• Keywords
• UV absorption spectra, fluorescent probes, molecular dynamics simulations, time‐dependent density functional theory,
• MeSH
• Fluorescent Dyes * chemistry   MeSH
• Peptides * chemistry   MeSH
• Rhodamines * chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Spectrophotometry, Ultraviolet MeSH
• Density Functional Theory * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fluorescent Dyes * MeSH
• Peptides * MeSH
• Rhodamines * MeSH
• tetramethylrhodamine MeSH Browser

Fluidity of lipid membranes is known to play an important role in the functioning of living organisms. The fluorescent probe Laurdan embedded in a lipid membrane is typically used to assess the fluidity state of lipid bilayers by utilizing the sensitivity of Laurdan emission to the properties of its lipid environment. In particular, Laurdan fluorescence is sensitive to gel vs liquid⁻crystalline phases of lipids, which is demonstrated in different emission of the dye in these two phases. Still, the exact mechanism of the environment effects on Laurdan emission is not understood. Herein, we utilize dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers, which at room temperature represent gel and liquid⁻crystalline phases, respectively. We simulate absorption and emission spectra of Laurdan in both DOPC and DPPC bilayers with quantum chemical and classical molecular dynamics methods. We demonstrate that Laurdan is incorporated in heterogeneous fashion in both DOPC and DPPC bilayers, and that its fluorescence depends on the details of this embedding.

• Keywords
• DFT, Laurdan, TDDFT, classical molecular dynamics, fluorescence,
• MeSH
• 1,2-Dipalmitoylphosphatidylcholine chemistry   MeSH
• 2-Naphthylamine analogs & derivatives   chemistry   MeSH
• Models, Chemical * MeSH
• Fluorescence MeSH
• Phosphatidylcholines chemistry   MeSH
• Quantum Theory MeSH
• Laurates chemistry   MeSH
• Lipid Bilayers chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 1,2-Dipalmitoylphosphatidylcholine MeSH
• 1,2-oleoylphosphatidylcholine MeSH Browser
• 2-Naphthylamine MeSH
• Phosphatidylcholines MeSH
• Laurates MeSH
• laurdan MeSH Browser
• Lipid Bilayers MeSH

A nucleoside bearing a solvatochromic push-pull fluorene fluorophore (dCFL ) was designed and synthesized by the Sonogashira coupling of alkyne-linked fluorene 8 with 5-iodo-2'-deoxycytidine. The fluorene building block 8 and labeled nucleoside dCFL exerted bright fluorescence with significant solvatochromic effect providing emission maxima ranging from 421 to 544 nm and high quantum yields even in highly polar solvents, including water. The solvatochromism of 8 was studied by DFT and ADC(2) calculations to show that, depending on the polarity of the solvent, emission either from the planar or the twisted conformation of the excited state can occur. The nucleoside was converted to its triphosphate variant dCFLTP which was found to be a good substrate for DNA polymerases suitable for the enzymatic synthesis of oligonucleotide or DNA probes by primer extension or PCR. The fluorene-linked DNA can be used as fluorescent probes for DNA-protein (p53) or DNA-lipid interactions, exerting significant color changes visible even to the naked eye. They also appear to be suitable for time-dependent fluorescence shift studies on DNA, yielding information on DNA hydration and dynamics.

• Publication type
• Journal Article MeSH

Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes because the molecular interactions taking place in lipid membranes frequently occur on the same timescale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity, and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how time-resolved fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (steady-state) approach will be presented in this review: 1), determination of membrane polarity and mobility using time-dependent spectral shifts; 2), identification of submicroscopic domains by fluorescence lifetime imaging microscopy; 3), elucidation of membrane leakage mechanisms from dye self-quenching assays; and 4), evaluation of nanodomain sizes by time-resolved Förster resonance energy transfer measurements.

• MeSH
• Fluorescent Dyes chemistry   MeSH
• Microscopy, Fluorescence methods   MeSH
• Kinetics MeSH
• Lipid Bilayers chemistry   MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Lipid Bilayers MeSH