Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations

Ablinger, Elisabeth; Leitgeb, Stefan; Zimmer, Andreas

doi:10.1016/j.ijpharm.2012.11.035

Cited by 29 publications

(19 citation statements)

References 25 publications

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“…[4][5][6] We confirmed that at the typical concentration of 5×, SYPRO Orange is not suitable for use for DSF measurements of surfactant-containing formulations. This can be seen in the SYPRO Orange DSF profile of mAb1 at 0.5 mg/ mL in a formulation that contains 0.04% (w/v) Tween 80 (Fig.…”

Section: Comparison Of Sypro Orange and Daspmi Dsf For Use In Screenisupporting

confidence: 59%

Simultaneous High-Throughput Conformational and Colloidal Stability Screening Using a Fluorescent Molecular Rotor Dye, 4-(4-(Dimethylamino)styryl)-N-Methylpyridinium Iodide (DASPMI)

et al. 2016

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Technologies to improve the throughput for screening protein formulations are continuously evolving. The purpose of this article is to highlight novel applications of a molecular rotor dye, 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DASPMI) in screening for the conformational stability, colloidal stability, and subtle pretransition dynamics of protein structures during early formulation development. The measurement of the apparent unfolding temperature (Tm) for a monoclonal antibody in the presence of Tween 80 was conducted and data were compared to the results of differential scanning calorimetry (DSC) measurements. Additionally, measuring the fluorescence intensity of DASPMI as a function of protein concentration shows consistent correlation to the diffusion interaction parameter (kD) for two distinct monoclonal antibody formulations measured by DLS. Lastly, due to the sensitivity of the molecular rotor dye to changes in microviscosity (ηmicro), subtle pretransition dynamics were discernable for two monoclonal antibody formulations that correlate with findings by red-edge excitation shift (REES) experiments. This novel application of molecular rotor dyes offers a valuable and promising approach for streamlining the early formulation development process due to low material consumption and rapid analysis time in a 96-well plate format.

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Section: Comparison Of Sypro Orange and Daspmi Dsf For Use In Screenisupporting

confidence: 59%

Simultaneous High-Throughput Conformational and Colloidal Stability Screening Using a Fluorescent Molecular Rotor Dye, 4-(4-(Dimethylamino)styryl)-N-Methylpyridinium Iodide (DASPMI)

et al. 2016

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“…Below pH 6, particles start to aggregate when they are in solution and our AFM and QCM-D experiments on adsorbed particles reveal major structural changes. We performed thermal denaturation experiments with Differential Scanning Fluorimetry (DSF) to further investigate the effect of pH on the stability of the particles ( Figure S1 ) 32 33 . When pH was changed from 7.5 to 6.0, the melting temperature (T m ) was reduced by only 1.5 °C.…”

Section: Resultsmentioning

confidence: 99%

Decrease in pH destabilizes individual vault nanocages by weakening the inter-protein lateral interaction

Llauró

Guerra

Kant

et al. 2016

Sci Rep

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Vault particles are naturally occurring proteinaceous cages with promising application as molecular containers. The use of vaults as functional transporters requires a profound understanding of their structural stability to guarantee the protection and controlled payload delivery. Previous results performed with bulk techniques or at non-physiological conditions have suggested pH as a parameter to control vault dynamics. Here we use Atomic Force Microscopy (AFM) to monitor the structural evolution of individual vault particles while changing the pH in real time. Our experiments show that decreasing the pH of the solution destabilize the barrel region, the central part of vault particles, and leads to the aggregation of the cages. Additional analyses using Quartz-Crystal Microbalance (QCM) and Differential Scanning Fluorimetry (DSF) are consistent with our single molecule AFM experiments. The observed topographical defects suggest that low pH weakens the bonds between adjacent proteins. We hypothesize that the observed effects are related to the strong polar character of the protein-protein lateral interactions. Overall, our study unveils the mechanism for the influence of a biologically relevant range of pHs on the stability and dynamics of vault particles.

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“…To further confirm the interaction of AtEXO70A1 with ES2 and ES2-14, we used another biochemical assay called differential scanning fluorimetry (DSF; Niesen et al, 2007), which has been commonly used to detect the interaction between small molecules and protein ligands (Ablinger et al, 2013;Baud et al, 2014;Athuluri-Divakar et al, 2016). The DSF assay is based on the theory that when a small-molecule ligand binds to the target protein, it could stabilize the protein and make the protein more tolerant to denaturation caused .…”

Section: Es2-14 Directly Interacts With Atexo70a1mentioning

confidence: 99%

Endosidin2-14 Targets the Exocyst Complex in Plants and Fungal Pathogens to Inhibit Exocytosis

Huang

et al. 2019

Plant Physiol.

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The evolutionarily conserved octameric exocyst complex tethers secretory vesicles to the site of membrane fusion during exocytosis. The plant exocyst complex functions in cell wall biosynthesis, polarized growth, stress responses, and hormone signaling. In fungal pathogens, the exocyst complex is required for growth, development, and pathogenesis. Endosidin2 (ES2) is known to inhibit exocytosis in plant and mammalian cells by targeting the EXO70 subunit of the exocyst complex. Here we show that an analog of ES2, ES2-14, targets plant and two fungal EXO70s. A lower dosage of ES2-14 than of ES2 is required to inhibit plant growth, plant exocytic trafficking, and fungal growth. ES2-14 treatments inhibit appressorium formation and reduce lesion sizes caused by Magnaporthe oryzae. Inhibition of EXO70 by ES2-14 in Botrytis cinerea also reduces its virulence in Arabidopsis (Arabidopsis thaliana). Interestingly, ES2-14 did not affect EXO70 localization or transferrin recycling in mammalian cells. Overall, our results indicate that a minor change in ES2 affects its specificity in targeting EXO70s in different organisms and they demonstrate the potential of using ES2-14 to study the mechanisms of plant and fungal exocytosis and the roles of exocytosis in fungus-plant interactions.

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Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations

Cited by 29 publications

References 25 publications

Simultaneous High-Throughput Conformational and Colloidal Stability Screening Using a Fluorescent Molecular Rotor Dye, 4-(4-(Dimethylamino)styryl)-N-Methylpyridinium Iodide (DASPMI)

Simultaneous High-Throughput Conformational and Colloidal Stability Screening Using a Fluorescent Molecular Rotor Dye, 4-(4-(Dimethylamino)styryl)-N-Methylpyridinium Iodide (DASPMI)

Decrease in pH destabilizes individual vault nanocages by weakening the inter-protein lateral interaction

Endosidin2-14 Targets the Exocyst Complex in Plants and Fungal Pathogens to Inhibit Exocytosis

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