Synthesis and Characterization of a New Bifunctionalized, Fluorescent, and Amphiphilic Molecule for Recruiting SH‐Containing Molecules to Membranes

Abstract

This study describes the synthesis and characterization of an amphiphilic construct intended to recruit SH-containing molecules to membranes. The construct consists of 1) an aliphatic chain to enable anchoring within membranes, 2) a maleimide moiety to react with the sulfhydryl group of a soluble (bio)molecule, and 3) a fluorescence moiety to allow the construct to be followed by fluorescence spectroscopy and microscopy. It is shown that the construct can be incorporated into preformed membranes, thus allowing application of the approach with biological membranes. The close proximity between the fluorophore and the maleimide moiety within the construct causes fluorescence quenching. This allows monitoring of the reaction with SH-containing molecules by measurement of increases in fluorescence intensity and lifetime. Notably, the construct distributes into laterally ordered membrane domains of lipid vesicles, which is probably triggered by the length of its membrane anchor. The advantages of the new construct can be employed for several biological, biotechnological, and medicinal applications.

Antibody Binding at the Liposome–Water Interface: A FRET Investigation toward a Liposome-Based Assay

“…According to Figure 1 (left), before the reaction with DTT, the fluorescence intensity of mm239 dye (in liposomes) was very weak due to the quenching effect of the maleimide group, which is a result of a photoinduced electron transfer. 12 After reaction with DTT, the fluorescence intensity increased significantly by a factor of about eight.…”

“…Some spectroscopic properties of the mm239-DTT in DMSO as well as immobilized in liposomes are shown in Table 1 (for mm239 only and in other solvents see ref (12)). The DBD derivative is characterized by a large Stokes shift (Δλ ∼ 90 ± 5 nm) and long fluorescence lifetime (τ ∼ 15 ns) in both samples.…”

“…Previously, the incorporation of mm239 into GUV membranes has been shown indirectly by fluorescence microscopy. 12 Here, additional fluorescence depolarization experiments were performed to monitor the incorporation of dyes (and the conjugation of antibodies to liposomes, vide infra) directly. A full depolarization of the fluorescence was found in DMSO, and subsequently the steady-state fluorescence anisotropy r was calculated as zero.…”

“…Due to the presence of a maleimide moiety, further biofunctionalization, e.g., to proteins, was successfully carried out. 12…”

Advances in the development of fluorescence probes for cell plasma membrane imaging

“…for SH-containing molecules (e.g. Cys) detection [233]. Probe 1 exhibited weak emission due to the PET quenching mechanisms, while the emission was switched "ON" after reacting of maleimide with biothiols.…”