Understanding the avidin-biotin binding based on polarized protein-specific charge

In this study, charge updating schemes based on the local polarized protein-specific charge (LPPC) were introduced to vary the atomic charges of the biotin molecule and the residues in close...

An in silico and in vitro pipeline for the rapid screening of helicase modulators

To evaluate the potency of potential helicase modulators, we developed an assay of helicase enzyme activity. Using a DNA or RNA biotin labelled oligonucleotide and after the addition of a recombinant helicase, the nucleic acid unwinds, causing the emission of luminescence, which is quantified with a particular antibody. In our assay, one of the DNA oligos was biotinylated, while the other was labelled with digoxygenin (DIG), both in their 5’ termini. The biotin molecule immobilises the DNA duplex on a neutravidin-coated plate and the helicase activity is measured through the unwinding of DNA, due to ATP activation. The subsequent release of DIG-labelled oligos results in a luminescence signal measured with a chemiluminescence antibody. Our goal was to provide a high throughput screening method for potential helicase inhibitors. The method described in this paper has been demonstrated to be fast, easy and reproducible and doesn’t use radiochemicals.

An in silico and in vitro pipeline for the rapid screening of helicase modulators

To evaluate the potency of potential helicase modulators, we developed an assay of helicase enzyme activity. Using a DNA or RNA biotin labelled oligonucleotide and after the addition of a recombinant helicase, the nucleic acid unwinds, causing the emission of luminescence, which is quantified with a particular antibody. In our assay, one of the DNA oligos was biotinylated, while the other was labelled with digoxygenin (DIG), both in their 5’ termini. The biotin molecule immobilises the DNA duplex on a neutravidin-coated plate and the helicase activity is measured through the unwinding of DNA, due to ATP activation. The subsequent release of DIG-labelled oligos results in a luminescence signal measured with a chemiluminescence antibody. Our goal was to provide a high throughput screening method for potential helicase inhibitors. The method described in this paper has been demonstrated to be fast, easy and reproducible and doesn’t use radiochemicals.

Dynamics of a Form-Fitting Protein in a Nanopore: Avidin in ClyA

ABSTRACTWe probe the molecular dynamics of a protein, avidin, as it is captured and trapped in a nanopore, ClyA, with time-resolved single-molecule electrical conductance measurements, and we present a method for visualizing this process from the data. The case of avidin in ClyA has rich time-dependent conductance spectra of discrete levels that correlate with different configurations of the protein in the pore. One is very long-lasting, stable and noise-free, and portends the use of this system as a platform for more general studies of proteins and other molecules, where avidin acts as a shuttle that ferries analytes into the pore for probing. We demonstrate this by the sensitive detection of a biotin molecule attached to avidin captured by the pore. We also present an approach to determining the nanopore size based on a 3D printed model of the pore.

Biotin transport in microvillous membrane vesicles, cultured trophoblasts, and isolated perfused human placenta

Biotin, essential for normal fetal growth and development, must be transported across the placenta to reach the fetus. This study evaluated placental transport of biotin using microvillous membrane vesicles (MMV), cultured trophoblasts, and isolated perfused cotyledon. Biotin uptake in MMV was stimulated by an inward Na+ gradient. In the presence of Na+, maximal stimulation was observed with Cl-, among various anions. Biotin uptake required 1 Na+ per biotin molecule. Kinetic analysis in MMV showed saturable transport with a Michaelis constant (Km) of 26.1 +/- 2.9 microM. Increases in membrane potential did not alter biotin uptake. Biotin uptake by cultured trophoblasts was also stimulated in the presence of Na+ and was saturable (Km = 7.0 +/- 1.5 microM). In the perfused placental cotyledon, maternal-to-fetal (M-to-F) biotin transfer was not saturable. However, biotin transfer in the M-to-F direction was significantly greater than the reverse. When the fetal circulation was closed to allow accumulation, an F/M ratio of only 1.056:1 was achieved. Tissue analysis of biotin contents suggested an active accumulation within the placental compartment. This study demonstrates that biotin is actively transported into the placenta, across the microvillous membrane, and released into the fetal compartment at a slower rate.

Interaction of Proteins with Functional Monolayers: Recognition, 2D-Crystallization and Function

The interaction of molecular self organization and molecular recognition leads to the formation of functional supramolecular systems (see Figure 1). They combine order and mobility and their function is based on their organization.’ These fascinating phenomena - and the living cell is only the most perfect example - can help to mimic biomembrane processes as well as to develop new materials. The interaction of proteins with biomembranes is of fundamental relevance in many fields of biochemistry and medicine. Model biomembranes offer the possibility to simulate such naturally occuring processes. The monolayer technique in particular has proven to be an ideal tool to investigate the interaction of water soluble proteins with model membranes. Unspecific adsorption of proteins at charged lipid monolayers, specific recognition of ligand molecules by proteins and enzyme binding followed by enzyme action may lead to highly oriented protein domains and even to 2D-crystallization (see Figure 2).Due to its high binding constant (Kd = 10−15 mol-1), the biotin/streptavidin system is an ideal tool to investigate the specific interaction between a membrane incorporated receptor and a protein. Streptavidin is a protein which comprises four identical subunits, each binding one biotin molecule, and is well known from affinity chromatography. Biotin lipids with biotinylated headgroups have been synthesized to act as the membrane-incorporated receptors. Injection of fluorescent-labeled streptavidin underneath a monolayer of biotin lipids in the gas-analogue state results in formation of regular H-shaped optically anisotropic two-dimensional streptavidin domains. The crystallinity of these two-dimensional domains was determined by electron crystallography, revealing diffraction patterns with a resolution of 1.5 nm. The match of the electron density map thus obtained and the X-ray crystal structure of streptavidin is remarkably close, showing that two binding sites are still free and face away from the lipid layer, exposed to the aqueous solution. This offers the possibility of building up protein multilayer structures - even a second crystalline protein layer is imaginable, using the first streptavidin layer as a matrix for the binding of other biotinylated proteins.