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1. Biotinylation as a tool to enhance the uptake of small molecules in Gram-negative bacteria

   https://doi.org/10.1371/journal.pone.0260023  

   Pandeya, Ankit; Yang, Ling; Alegun, Olaniyi; Karunasena, Chamikara; Risko, Chad; Li, Zhenyu; Wei, Yinan (November 2021, PLOS ONE)

   van Veen, Hendrik W. (Ed.)

   Antibiotic resistance is a major public health concern. The shrinking selection of effective antibiotics and lack of new development is making the situation worse. Gram-negative bacteria more specifically pose serious threat because of their double layered cell envelope and effective efflux systems, which is a challenge for drugs to penetrate. One promising approach to breach this barrier is the “Trojan horse strategy”. In this technique, an antibiotic molecule is conjugated with a nutrient molecule that helps the antibiotic to enter the cell through dedicated transporters for the nutrient. Here, we explored the approach using biotin conjugation with a florescent molecule Atto565 to determine if biotinylation enhances accumulation. Biotin is an essential vitamin for bacteria and is obtained through either synthesis or uptake from the environment. We found that biotinylation enhanced accumulation of Atto565 in E . coli . However, the enhancement did not seem to be due to uptake through biotin transporters since the presence of free biotin had no observable impact on accumulation. Accumulated compound was mostly in the periplasm, as determined by cell fractionation studies. This was further confirmed through the observation that expression of streptavidin in the periplasm specifically enhanced the accumulation of biotinylated Atto565. This enhancement was not observed when streptavidin was expressed in the cytoplasm indicating no significant distribution of the compound inside the cytoplasm. Using gene knockout strains, plasmid complementation and mutagenesis studies we demonstrated that biotinylation made the compound a better passenger through OmpC, an outer membrane porin. Density functional theory (DFT)-based evaluation of the three-dimensional geometries showed that biotinylation did not directly stabilize the conformation of the compound to make it favorable for the entry through a pore. Further studies including molecular dynamics simulations are necessary to determine the possible mechanisms of enhanced accumulation of the biotinylated Atto565. 

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2. Loading characteristics of streptavidin on polypropylene capillary channeled polymer fibers and capture performance towards biotinylated proteins

   https://doi.org/10.1007/s00216-023-04948-5  

   Islam, Md_Khalid Bin; Kenneth_Marcus, R (November 2023, Analytical and Bioanalytical Chemistry)

   continuing emphasis. Polypropylene (PP) capillary-channeled polymer (C-CP) fiber columns are modified with the biotin- binding protein streptavidin (SAV) to capture biotinylated proteins. The loading characteristics of SAV on fiber supports were determined using breakthrough curves and frontal analysis. Based on adsorption data, a 3-min on-column loading at a flow rate of 0.5 mL min−1 (295.2 cm h−1) with a SAV feed concentration of 0.5 mg mL−1 produces a SAV loading capacity of 1.4 mg g−1 fiber. SAV has an incredibly high affinity for the small-molecule biotin (10−14 M), such that this binding relationship can be exploited by labeling a target protein with biotin via an Avi-tag. To evaluate the capture of the biotinylated proteins on the modified PP surface, the biotinylated versions of bovine serum albumin (b-BSA) and green fluorescent protein (b-GFP) were utilized as probe species. The loading buffer composition and flow rate were optimized towards protein capture. The non-ionic detergent Tween-20 was added to the deposition solutions to minimize non-specific binding. Values of 0.25–0.50% (v/v) Tween-20 in PBS exhibited better capture efficiency, while minimizing the non-specific binding for b-BSA and b-GFP, respectively. The C-CP fiber platform has the potential to provide a fast and low-cost method to capture targeted proteins for applications including protein purification or pull-down assays. 

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3. Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein

   https://doi.org/10.1073/pnas.2109169119  

   Gaffney, Kristen_A; Guo, Ruiqiong; Bridges, Michael_D; Muhammednazaar, Shaima; Chen, Daoyang; Kim, Miyeon; Yang, Zhongyu; Schilmiller, Anthony_L; Faruk, Nabil_F; Peng, Xiangda; et al (December 2021, Proceedings of the National Academy of Sciences)

   Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG ofEscherichia coli(E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron–electron resonance spectroscopy. These data, along with ourUpsidesimulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition.E. coli’s lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes. 

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4. An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET

   https://doi.org/10.7554/eLife.70236  

   Zagotta, William N; Sim, Brandon S; Nhim, Anthony K; Raza, Marium M; Evans, Eric GB; Venkatesh, Yarra; Jones, Chloe M; Mehl, Ryan A; Petersson, E James; Gordon, Sharona E (October 2021, eLife)

   With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions. 

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5. Cell type-specific biotin labeling in vivo resolves regional neuronal and astrocyte proteomic differences in mouse brain

   https://doi.org/10.1038/s41467-022-30623-x  

   Rayaprolu, Sruti; Bitarafan, Sara; Santiago, Juliet V.; Betarbet, Ranjita; Sunna, Sydney; Cheng, Lihong; Xiao, Hailian; Nelson, Ruth S.; Kumar, Prateek; Bagchi, Pritha; et al (May 2022, Nature Communications)

   Abstract Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states. 

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