Quantifying the Interaction of Phosphite with ABC Transporters: MicroScale Thermophoresis and a Novel His-Tag Labeling Approach

Bartoschik, Tanja; Gupta, Amit J.; Kern, Beate; Hitchcock, Andrew; Adams, Nathan B. P.; Tschammer, Nuška

doi:10.1007/978-1-0716-0724-4_2

Cited by 7 publications

(8 citation statements)

References 27 publications

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“…Moreover, the addition of DDM after SEC improves the homogeneity of the samples without compromising the protein aggregation state. It is noteworthy that the experimental procedure and buffers we used are in line with previous investigations [8,14,17,20,21], so extensive protein aggregation or misfolding were not noticeable in our experiments. Even though the DDM supplementation results in c(s) distributions overlapping with a high concentration of DDM, it still remains to be experimentally determined whether TmrA possesses similar activity.…”

Section: Discussionsupporting

confidence: 84%

“…These detergents are mild nonionic detergents and help maintain membrane protein conformations. In previous studies, with respect to ABC transporters, purification workflow has been optimized, and DDM was the detergent of choice [11,[16][17][18][19][20][21]. For example, in the study of BmrA purified from B. subtilis, a number of nonionic detergents and various concentrations of DDM were investigated for the optimal purification of BmrA, and BmrA was purified in a functional form in DDM [9].…”

Section: Introductionmentioning

confidence: 99%

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Optimization of Membrane Protein TmrA Purification Procedure Guided by Analytical Ultracentrifugation

Chu

Sheng

et al. 2021

Membranes

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Membrane proteins are involved in various cellular processes. However, purification of membrane proteins has long been a challenging task, as membrane protein stability in detergent is the bottleneck for purification and subsequent analyses. Therefore, the optimization of detergent conditions is critical for the preparation of membrane proteins. Here, we utilize analytical ultracentrifugation (AUC) to examine the effects of different detergents (OG, Triton X-100, DDM), detergent concentrations, and detergent supplementation on the behavior of membrane protein TmrA. Our results suggest that DDM is more suitable for the purification of TmrA compared with OG and TritonX-100; a high concentration of DDM yields a more homogeneous protein aggregation state; supplementing TmrA purified with a low DDM concentration with DDM maintains the protein homogeneity and aggregation state, and may serve as a practical and cost-effective strategy for membrane protein purification.

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Section: Discussionsupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Optimization of Membrane Protein TmrA Purification Procedure Guided by Analytical Ultracentrifugation

Chu

Sheng

et al. 2021

Membranes

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“…This allows an affinity determination of a protein–ligand interaction by observing fluorescence changes. ,, Besides thermophoresis, other effects can cause these changes, e.g., temperature differences and changes in the local environment of the fluorophore. , The advantages of this method are certainly its sensitivity and scalability, but also the fact that screenings can be performed directly with cell lysates or blood sera, which makes it a powerful method for drug discovery. ,,,− Determining binding affinity by MST, however, comes with some inherent problems. In most cases, the protein of interest must be fluorescently labeled, either in a covalent manner, by thiol or amide coupling, or in a non-covalent manner, by His-tag labeling with a fluorescent dye. , Direct measuring of protein–ligand interactions by MST only shows binding events but cannot reveal if the detected binding is competitive with respect to the active site of the protein, or if the ligand binding occurs on an allosteric site. It can also be very challenging to reach saturation conditions for the bound protein–ligand complex, especially if ligands only have binding affinities in the low single-digit micromolar range like the currently known inhibitors of DNMT2.…”

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confidence: 99%

An Optimized Microscale Thermophoresis Method for High-Throughput Screening of DNA Methyltransferase 2 Ligands

Zimmermann

Schwickert

Meidner

et al. 2022

ACS Pharmacol. Transl. Sci.

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Developing methyltransferase inhibitors is challenging, since most of the currently used assays are time-consuming and cost-intensive. Therefore, efficient, fast, and reliable methods for screenings and affinity determinations are of utmost importance. Starting from a literature-known fluorescent S-adenosylhomocysteine derivative, 5-FAM-triazolyl-adenosyl-Dab, developed for a fluorescence polarization assay to investigate the histone methyltransferase mixed-lineage leukemia 1, we herein describe the applicability of this compound as a fluorescent tracer for the investigation of DNA-methyltransferase 2 (DNMT2), a human RNA methyltransferase. Based on these findings, we established a microscale thermophoresis (MST) assay for DNMT2. This displacement assay can circumvent various problems inherent to this method. Furthermore, we optimized a screening method via MST which even indicates if the detected binding is competitive and gives the opportunity to estimate the potency of a ligand, both of which are not possible with a direct binding assay.

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“…Non-covalent labeling has the advantage of being less disruptive to protein structure and is often the labeling strategy of choice for unstable or challenging targets such as membrane proteins. 36 …”

Section: Resultsmentioning

confidence: 99%

A New Spectral Shift-Based Method to Characterize Molecular Interactions

Langer

Bartoschik

Cehlár

et al. 2022

ASSAY and Drug Development Technologies

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There are many fluorescence-based applications that can be used to characterize molecular interactions. However, available methods often depend on site-specific labeling techniques or binding-induced changes in conformation or size of the probed target molecule. To overcome these limitations, we applied a ratiometric dual-emission approach that quantifies ligand-induced spectral shifts with sub-nanometer sensitivity. The use of environment-sensitive near-infrared dyes with the method we describe enables affinity measurements and thermodynamic characterization without the explicit need for site-specific labeling or ligand-induced conformational changes. We demonstrate that in-solution spectral shift measurements enable precise characterization of molecular interactions for a variety of biomolecules, including proteins, antibodies, and nucleic acids. Thereby, the described method is not limited to a subset of molecules since even the most challenging samples of research and drug discovery projects like membrane proteins and intrinsically disordered proteins can be analyzed.

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Quantifying the Interaction of Phosphite with ABC Transporters: MicroScale Thermophoresis and a Novel His-Tag Labeling Approach

Cited by 7 publications

References 27 publications

Optimization of Membrane Protein TmrA Purification Procedure Guided by Analytical Ultracentrifugation

Optimization of Membrane Protein TmrA Purification Procedure Guided by Analytical Ultracentrifugation

An Optimized Microscale Thermophoresis Method for High-Throughput Screening of DNA Methyltransferase 2 Ligands

A New Spectral Shift-Based Method to Characterize Molecular Interactions

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