Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM1 and GM1-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM1 positive cooperativity. Surprisingly, we demonstrated fucosyl-GM1 has approximately 7 times higher CTB binding capacity than GM1. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM1 caused the increased binding capacity. This was unintuitive, as GM1 exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM1 and GM2, we observed the mild binding avidity fucosyl-GM1 activated GM2 receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM2 receptors have negligible binding avidity in pure GM2 bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.
GM1 has generally been considered as the major receptor that binds to cholera toxin subunit B (CTB) due to its low dissociation constant. However, using a unique nanocube sensor technology, we have shown that CTB can also bind to other glycolipid receptors, fucosyl-GM1 and GD1b. Additionally, we have demonstrated that GM2 can contribute to CTB binding if present in a glycolipid mixture with a strongly binding receptor (GM1/fucosyl-GM1/GD1b). This hetero-multivalent binding result was unintuitive because the interaction between CTB and pure GM2 is negligible. We hypothesized that the reduced dimensionality of CTB-GM2 binding events is a major cause of the observed CTB binding enhancement. Once CTB has attached to a strong receptor, subsequent binding events are confined to a 2D membrane surface. Therefore, even a weak GM2 receptor could now participate in second or higher binding events because its surface reaction rate can be up to 104 times higher than the bulk reaction rate. To test this hypothesis, we altered the surface reaction rate by modulating the fluidity and heterogeneity of the model membrane. Decreasing membrane fluidity reduced the binding cooperativity between GM2 and a strong receptor. Our findings indicated a new protein-receptor binding assay, that can mimic complex cell membrane environment more accurately, is required to explore the inherent hetero-multivalency of the cell membrane. We have thus developed a new membrane perturbation protocol to efficiently screen receptor candidates involved in hetero-multivalent protein binding.
Surface-enhanced Raman scattering (SERS) is a powerful analytical tool which enables the detection and identification of analytes adsorbed on nanostructured noble metals. However, SERS analysis of complex mixtures can be challenging due to spectral overlap and interference. In this report, we demonstrate a method to simplify the identification of mixed-analyte samples by coupling SERS detection with chromatographic separation on a nanoplasmonic paper substrate. This "nanopaper" substrate is a silver coated glass microfiber filter paper which possesses large SERS enhancement and can serve as a stationary phase in paper chromatography. Nanopaper is easily synthesized using the silver mirror reaction, making it a highly accessible technology. Nanopaper was successfully used as a combined paper chromatography-SERS (PC-SERS) substrate in the separation and identification of mixed organic dyes. It was further employed to separate and identify lycopene and β-carotene in commercial food products, demonstrating the versatility and utility of nanopaper in the identification of complex mixtures.
Surface-enhanced Raman spectroscopy (SERS) is a powerful tool with high potential for multiplexed detection of dilute analytes. However, quantitative SERS of kinetic assays can be difficult due to the variation in enhancement factors caused by changing reaction conditions. We report a method for quantitative SERS kinetic analysis using colloidal Ag-Au core-shell nanocubes (Ag@AuNCs) as the SERS substrate. This substrate is mass producible, possesses large SERS enhancement, and is resistant to degradation in most environments. The SERS enhancement of the Ag@AuNCs was evaluated both experimentally and computationally. Quantitation was achieved by covalently attaching a non-reactive internal standard (IS) to substrate surfaces and normalizing SERS spectra to the IS signal. We demonstrated that IS normalization corrects for temporal variations in enhancement factor and particle concentration. Quantitation was demonstrated by monitoring the base-catalyzed aldol condensation of surface-bound 4-(methylthio)benzaldehyde with free acetone. The kinetic model of this reaction was fitted to IS normalized SERS data, resulting in kinetic parameters that agreed well with published values. This SERS platform is a robust and sensitive method for quantitative analysis of kinetic assays, with potential applications in many fields.
Adaptive laboratory evolution (ALE) is a powerful tool used to increase strain fitness in the presence of environmental stressors. If production and strain fitness can be coupled, ALE can be used to increase product formation. In earlier work, carotenoids hyperproducing mutants were obtained using an ALE strategy. Here, de novo mutations were identified in hyperproducers, and reconstructed mutants were explored to determine the exact impact of each mutation on production and tolerance. A single mutation in YMRCTy1-3 conferred increased carotenoid production, and when combined with other beneficial mutations led to further increased β-carotene production. Findings also suggest that the ALE strategy selected for mutations that confer increased carotenoid production as primary phenotype. Raman spectroscopy analysis and total lipid quantification revealed positive correlation between increased lipid content and increased β-carotene production. Finally, we demonstrated that the best combinations of mutations identified for β-carotene production were also beneficial for production of lycopene.
Tuberculosis (TB) is among the greatest public health and safety concerns in the 21st century Mycobacterium tuberculosis, which causes TB, infects alveolar macrophages and uses these cells as one of its primary sites of replication. The current TB treatment regimen, which consists of chemotherapy involving a combination of 3-4 antimicrobials for a duration of 6-12 months, is marked with significant side effects, toxicity, and poor compliance. Targeted drug delivery offers a strategy that can overcome many of the problems of current TB treatment by specifically targeting infected macrophages. Recent advances in nanotechnology and material science have opened an avenue to explore drug carriers that actively and passively target macrophages. This approach can increase the drug penetration into macrophages by using ligands on the nanocarrier that interact with specific receptors for macrophages. This review encompasses the recent development of drug carriers specifically targeting macrophages actively and passively. Future directions and challenges associated with development of effective TB treatment are also discussed.
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