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. This method can be applied for PS content determination in protein formulations (≤100 mg/mL) without the need of a previous protein removal step. The 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate assay implemented in multi-well plate format is suitable for high-throughput concentration screening. It has a linear range from 0.00020% to 0.0025% (w/v) PS20, and the limits of detection and quantification were 0.00020% and 0.00055% (w/v), respectively. This assay is markedly more selective and shows no or lower interferences due to hydrophobic components (e.g., silicone oil) potentially present in finished products than the fluorescence micelle assay based on N-phenyl-1-naphthylamine. It also provides comparable results for the PS content in liquid chromatography with charged aerosol detection analysis with protein removal, providing a fast alternative.

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. This review article focuses on methods and strategies reported in the recent years for the analytical characterization of PSs, their degradants and other impurities within neat PS (i.e., PS raw materials), diluted PS solutions, as well as in biopharmaceutical formulations. The use of versatile and complementary methods applied in a systematic approach is crucial to understand the impact of the concentration, composition, and degradation of PSs on the quality of biopharmaceutical products.

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. Similar to surface plasmon resonance (SPR), the gold nanorods report changes in their protein surface coverage without the need for fluorescence labeling, a technique we refer to as NanoSPR. Comparing the dynamics for fluorescence labeled and unlabeled proteins, we find a reduction of the oscillation period by about 20%. The absence of photobleaching allows us to investigate Min proteins attaching and detaching from lipid coated gold nanorods with an unprecedented bandwidth of 100 ms time resolution and 1 h observation time. The long observation reveals small changes of the oscillation period over time. Averaging many cycles yields the precise wave profile that exhibits the four phases suggested in previous reports. Unexpected from previous fluorescence-based studies, we found an immobile static protein layer not dissociating during the oscillation cycle. Hence, NanoSPR is an attractive label-free real-time technique for the local investigation of molecular dynamics with high observation bandwidth. It gives access to systems, which cannot be fluorescently labeled, and resolves local dynamics that would average out over the sensor area used in conventional SPR.

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. We show that MinCDE drives the formation of waves of FtsZ polymers associated to bilayers by ZipA, which propagate as antiphase patterns with respect to those of Min as revealed by confocal fluorescence microscopy. The emergence of these FtsZ waves results from the displacement of FtsZ polymers from the vicinity of the membrane by MinCD, which efficiently competes with ZipA for the C-terminal region of FtsZ, a central hub for multiple interactions that are essential for division. The coupling between FtsZ polymers and Min is enhanced at higher surface densities of ZipA or in the presence of crowding agents that favor the accumulation of FtsZ polymers near the membrane. The association of FtsZ polymers to the membrane modifies the response of FtsZ to Min, and comigrating Min-FtsZ waves are observed when FtsZ is free in solution and not attached to the membrane by ZipA. Taken together, our findings show that the dynamic Min patterns modulate the spatial distribution of FtsZ polymers in controlled minimal membranes. We propose that ZipA plays an important role in mid-cell recruitment of FtsZ orchestrated by MinCDE.

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. However, there has been a marked discrepancy between the speed of propagation of Min protein waves in vitro, compared with the cellular system. A very plausible explanation is the different mobility of proteins on model membranes, compared with the inner membrane of bacteria. To quantitatively demonstrate how membrane diffusion influences Min wave propagation, we compared Min waves on SLBs with free-standing giant unilamellar vesicles (GUV) membranes which display higher fluidity. Intriguingly, the propagation velocity and wavelength on GUVs are three times higher than those reported on supported bilayers, but the wave period is conserved. This suggests that the shorter spatial period of the patterns in vivo might indeed be primarily explained by lower diffusion coefficients of proteins on the bacterial inner membrane.

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. Previous experimental work to reconstitute elements of the bacterial division machinery in these membrane-like systems is summarized. Future challenges towards reconstructing minimal divisome assemblies in giant vesicles as cytomimetic containers are discussed.

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. To test how the orientation of the monomers affects the curvature of the filaments on a surface, we performed site-directed mutagenesis on the E. coli FtsZ protein to insert cysteine residues at lateral locations to orient FtsZ on planar lipid bilayers. The E93C and S255C mutants were overproduced, purified, and found to be functionally active in solution, as well as being capable of sustaining cell division in vivo in complementation assays. Atomic force microscopy was used to observe the shape of the filament fibers formed on the surface. The FtsZ mutants were covalently linked to the lipids and could be polymerized on the bilayer surface in the presence of GTP. Unexpectedly, both mutants assembled into straight structures. E93C formed a well-defined lattice with monomers interacting at 60° and 120° angles, whereas S255C formed a more open array of straight thicker filament aggregates. These results indicate that filament curvature and bending are not fixed and that they can be modulated by the orientation of the monomers with respect to the membrane surface. As filament curvature has been associated with the force generation mechanism, these results point to a possible role of filament membrane attachment in lateral association and curvature, elements currently identified as relevant for force generation.

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. These developments, together with new imaging and membrane reconstitution technologies, have triggered the 'bottom-up' synthetic approach aiming at reconstructing bacterial division in the test tube, which is required to support conclusions derived from cellular and molecular analysis. Here, we describe recent advances in reconstituting Escherichia coli minimal systems able to reproduce essential functions, such as the initial steps of division (proto-ring assembly) and one of the main positioning mechanisms (Min oscillating system), and discuss future perspectives and experimental challenges.

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. Binding of FtsA to lipids and membranes is insensitive to ionic strength, indicating that a net contribution of hydrophobic interactions is involved in the association of FtsA to lipid/membrane structures.

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. We have found that (i) ClpB self-associates from monomers to form hexamers and higher-order oligomers that have been tentatively assigned to dodecamers, (ii) oligomer dissociation is not accompanied by modifications of the protein secondary structure, (iii) the M domain is engaged in intersubunit interactions that stabilize the protein hexamer, and (iv) the nucleotide-induced rearrangement of ClpB affects the protein oligomeric core, in addition to the proposed radial extension of the M domain. The difference in the stability of the ATP- and ADP-bound states [ΔΔG(ATP-ADP) = -10 kJ/mol] might explain how nucleotide exchange promotes the conformational change of the protein particle that drives its functional cycle.

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. In the presence of GTP analogs, FtsZ assembled inside the GUIMVs, forming a web of dense spots and fibers. Whereas isolated FtsA was found adsorbed to the inner face of GUIMVs, the addition of FtsZ together with GTP analogs resulted in its dislodgement and its association with the FtsZ fibers in the lumen, suggesting that the FtsA-membrane interaction can be modulated by FtsZ polymers. The use of this novel in vitro system to probe interactions between divisome components will help to determine the biological implications of these findings.

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. However, in the absence of Mg(2+) (with EDTA), the data are best explained by an equilibrium model in which only B, A(1), A(2), A(3), A(1)B, and A(2)B contribute significantly to scattering. The best-fit molecular weights of monomeric A and B are in good agreement with values calculated from amino acid composition and with values obtained from sedimentation equilibrium. The latter technique also confirmed the interaction between sZipA and GDP-FtsZ. Moreover, the association model that best describes the CG-SLS data is in qualitative agreement with the sedimentation data. From these results, it follows that the binding of sZipA to GDP-FtsZ is of moderate affinity and does not significantly affect the interactions between FtsZ monomers. Under the working conditions used, only one sZipA binds to FtsZ oligomers with a length of six at most. The observed behavior would be compatible with FtsZ fibrils being anchored in vivo to the bacterial inner plasma membrane by substoichiometric binding of membrane-bound ZipA.

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. This raised the question of how the cell can prevent their interaction and thereby the premature stabilization of FtsZ protofilaments in nondividing cells. Therefore, we studied the FtsZ-ZapA interaction at the physiological pH of 7.5 instead of pH 6.5 (the optimal pH for FtsZ polymerization), under conditions that stimulate protofilament formation (5 mM MgCl(2)) and under conditions that stimulate and stabilize protofilaments (10 mM MgCl(2)). Using pelleting, light scattering, and GTPase assays, it was found that stabilization and bundling of FtsZ polymers by ZapA was inversely correlated to the GTPase activity of FtsZ. As GTP hydrolysis is the rate-limiting factor for depolymerization of FtsZ, we propose that ZapA will only enhance the cooperativity of polymer association during the transition from helical filament to mid cell ring and will not stabilize the short single protofilaments in the cytoplasm. All thus far published in vitro data on the interaction between FtsZ and ZapA have been obtained with His-ZapA. We found that in our case the presence of a His tag fused to ZapA prevented the protein to complement a DeltazapA strain in vivo and that it affected the interaction between FtsZ and ZapA in vitro.

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. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOH-terminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The first coiled-coil complex is dimeric and undergoes concentration-dependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0 A crystallographic data. Both coiled-coils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation.

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. Agonistic compounds bound to TodS and induced gene expression in vivo. Toluene was a powerful agonist, but ortho-substitutions of toluene reduced or abolished in vivo responses, although TodS recognized o-xylene with high affinity. These compounds were called antagonists. We show that agonists and antagonists compete for binding to TodS both in vitro and in vivo. The failure of antagonists to induce gene expression in vivo correlated with their inability to stimulate TodS autophosphorylation in vitro. We propose intramolecular TodS signal transmission, not molecular recognition of compounds by TodS, to be the phenomenon that determines whether a given compound will lead to activation of expression of the tod genes. Molecular modeling identified residues F46, I74, F79, and I114 as being potentially involved in the binding of effector molecules. Alanine substitution mutants of these residues reduced affinities (2- to 345-fold) for both agonistic and antagonistic compounds. Our data indicate that determining the inhibitory activity of antagonists is a potentially fruitful alternative to design specific two-component system inhibitors for the development of new drugs to inhibit processes regulated by two-component systems.