Novel High-Throughput Assay for Polysorbate Quantification in Biopharmaceutical Products by Using the Fluorescent Dye DiI.

Polysorbates (PSs) are the most common surfactants in therapeutic protein formulations, and it is crucial to monitor their concentration along the life cycle of biopharmaceuticals. We developed a simple multi-well plate fluorescence-based assay for the rapid determination of PS20 and PS80 content in biopharmaceutical products. The method is based on the detection of the fluorescence emission intensity of the fluorescent dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate in the presence of PSs at concentrations below their critical micelle concentration.

Trends on Analytical Characterization of Polysorbates and Their Degradation Products in Biopharmaceutical Formulations.

Among many other applications, polysorbates (PSs) are used as the most common surfactants in biopharmaceutical products in particular to protect proteins against interfacial stress. Structural heterogeneity, presence of degradants and other impurities, and tendency for degradation are interrelated features found in commercial PSs with a direct impact on their functional properties in biopharmaceutical products. These pose a challenge for the analytical characterization of PSs at different stages of product development.

Single Particle Plasmon Sensors as Label-Free Technique To Monitor MinDE Protein Wave Propagation on Membranes.

We use individual gold nanorods as pointlike detectors for the intrinsic dynamics of an oscillating biological system. We chose the pattern forming MinDE protein system from Escherichia coli (E. coli), a prominent example for self-organized chemical oscillations of membrane-associated proteins that are involved in the bacterial cell division process.

FtsZ Polymers Tethered to the Membrane by ZipA Are Susceptible to Spatial Regulation by Min Waves.

Bacterial cell division is driven by an FtsZ ring in which the FtsZ protein localizes at mid-cell and recruits other proteins, forming a divisome. In Escherichia coli, the first molecular assembly of the divisome, the proto-ring, is formed by the association of FtsZ polymers to the cytoplasmic membrane through the membrane-tethering FtsA and ZipA proteins. The MinCDE system plays a major role in the site selection of the division ring because these proteins oscillate from pole to pole in such a way that the concentration of the FtsZ-ring inhibitor, MinC, is minimal at the cell center, thus favoring FtsZ assembly in this region.

Propagation of MinCDE waves on free-standing membranes.

As a spatial modulator of cytokinesis in Escherichia coli, the Min system cooperates with the nucleoid occlusion mechanism to target the divisome assembly towards mid-cell. Based on a reaction-diffusion mechanism powered by ATP (adenosine triphosphate) hydrolysis, the Min proteins propagate in waves on the cell membrane, resulting in oscillations between the cell poles, thus preventing the formation of the division ring everywhere but in the cell centre. The dynamic behaviour of Min proteins has been successfully reconstructed in vitro on supported lipid bilayers (SLBs), reproducing many of the features observed in the cell.

Giant vesicles: a powerful tool to reconstruct bacterial division assemblies in cell-like compartments.

The use of artificial lipid membranes, structured as giant unilamellar vesicles (GUVs), provides the opportunity to investigate membrane-associated biological processes under defined experimental conditions. Due to their large size, they are uniquely adapted to investigate the properties and organization (in time and space) of macromolecular complexes incorporated in the vesicle interior by imaging and micro-spectroscopic techniques. Experimental methods to produce giant vesicles and to encapsulate proteins inside them are here reviewed.

Polymorphism of FtsZ filaments on lipid surfaces: role of monomer orientation.

FtsZ is a bacterial cytoskeletal protein involved in cell division. It forms a ringlike structure that attaches to the membrane to complete bacterial division. It binds and hydrolyzes GTP, assembling into polymers in a GTP-dependent manner.

Towards a bottom-up reconstitution of bacterial cell division.

The components of the bacterial division machinery assemble to form a dynamic ring at mid-cell that drives cytokinesis. The nature of most division proteins and their assembly pathway is known. Our knowledge about the biochemical activities and protein interactions of some key division elements, including those responsible for correct ring positioning, has progressed considerably during the past decade.

Isolation, characterization and lipid-binding properties of the recalcitrant FtsA division protein from Escherichia coli.

We have obtained milligram amounts of highly pure Escherichia coli division protein FtsA from inclusion bodies with an optimized purification method that, by overcoming the reluctance of FtsA to be purified, surmounts a bottleneck for the analysis of the molecular basis of FtsA function. Purified FtsA is folded, mostly monomeric and interacts with lipids. The apparent affinity of FtsA binding to the inner membrane is ten-fold higher than to phospholipids, suggesting that inner membrane proteins could modulate FtsA-membrane interactions.

A quantitative analysis of the effect of nucleotides and the M domain on the association equilibrium of ClpB.

ClpB is a hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. The hexamer is a highly dynamic complex, able to reshuffle subunits. To further characterize the biological implications of the ClpB oligomerization state, the association equilibrium of the wild-type (wt) protein and of two deletion mutants, which lack part or the whole M domain, was quantitatively analyzed under different experimental conditions, using several biophysical [analytical ultracentrifugation, composition-gradient (CG) static light scattering, and circular dichroism] and biochemical (ATPase and chaperone activity) methods.

Reconstitution and organization of Escherichia coli proto-ring elements (FtsZ and FtsA) inside giant unilamellar vesicles obtained from bacterial inner membranes.

We have incorporated, for the first time, FtsZ and FtsA (the soluble proto-ring proteins from Escherichia coli) into bacterial giant unilamellar inner membrane vesicles (GUIMVs). Inside the vesicles, the structural organization and spatial distribution of fluorescently labeled FtsZ and FtsA were determined by confocal microscopy. We found that, in the presence of GDP, FtsZ was homogeneously distributed in the lumen of the vesicle.

Characterization of self-association and heteroassociation of bacterial cell division proteins FtsZ and ZipA in solution by composition gradient-static light scattering.

We have characterized the self-association of FtsZ in its GDP-bound state (GDP-FtsZ) and the heteroassociation of FtsZ and a soluble recombinant ZipA (sZipA) lacking the N-terminal transmembrane domain by means of composition gradient-static light scattering (CG-SLS) and by measurement of sedimentation equilibrium. CG-SLS experiments at high ionic strengths and in the presence of 5 mM Mg(2+) show that, while FtsZ self-associates in a noncooperative fashion, sZipA acts as a monomer. CG-SLS data obtained from mixtures of FtsZ (A) and sZipA (B) in the presence of Mg(2+) are quantitatively described by an equilibrium model that takes into account significant scattering contributions from B, A(1), A(2), A(3), A(4), A(5), A(6), A(1)B, A(2)B, A(3)B, and A(4)B.

The GTPase activity of Escherichia coli FtsZ determines the magnitude of the FtsZ polymer bundling by ZapA in vitro.

FtsZ polymerizes in a ring-like structure at mid cell to initiate cell division in Escherichia coli. The ring is stabilized by a number of proteins among which the widely conserved ZapA protein. Using antibodies against ZapA, we found surprisingly that the cellular concentration of ZapA is approximately equal to that of FtsZ.

The KCNQ1 (Kv7.1) COOH terminus, a multitiered scaffold for subunit assembly and protein interaction.

The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias.

Bacterial sensor kinase TodS interacts with agonistic and antagonistic signals.

The TodS/TodT two-component system controls expression of the toluene dioxygenase (TOD) pathway for the metabolism of toluene in Pseudomonas putida DOT-T1E. TodS is a sensor kinase that ultimately controls tod gene expression through its cognate response regulator, TodT. We used isothermal titration calorimetry to study the binding of different compounds to TodS and related these findings to their capacity to induce gene expression in vivo.