Protein-binding assays in biological liquids using microscale thermophoresis

Wienken, Christoph J.; Philipp, B.; Rothbauer, Ulrich; Braun, Dieter; Duhr, Stefan

doi:10.1038/ncomms1093

Cited by 978 publications

(902 citation statements)

References 36 publications

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“…Serial dilutions of the non-labelled GAPDH molecules (10 ml each) were mixed with 10 ml of the fluorescently labelled transferrin molecule. The final sample was loaded into Monolith NT.115 capillaries, followed by analysis with the Monolith NT.115 instrument (Nanotemper) 61 .…”

Section: Methodsmentioning

confidence: 99%

Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin

et al. 2014

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Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages.

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Section: Methodsmentioning

confidence: 99%

Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin

et al. 2014

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“…The detailed principle and theory have been reported before. [31] In the present study, it relies on the detection of the difference of fluorescence signal intensity caused by the directional movement of FITC label on the hIAPP [11][12][13][14][15][16][17][18][19][20] (FITClabeled hIAPP [11][12][13][14][15][16][17][18][19][20] ) along the temperature gradient in microenvironment. The affinities of molecular interactions were measured by titration experiments in the equilibrium state.…”

Section: Background Of Thermophoresismentioning

confidence: 99%

“…Rln(K d ) and 1/T was plotted in the inset of Figure 5 to obtain the thermodynamic parameters ΔH and ΔS on the basis of van't Hoff plot. [31] The linear regression equation was established and listed in Table 2, and the types of binding events were determined by the principles of thermodynamics. [30] The results showed that the interaction between hIAPP [11][12][13][14][15][16][17][18][19][20] and these lipopeptide inhibitors is all spontaneous exothermic processes because of the negative change in Gibbs free energy and enthalpy changes.…”

Section: Thermodynamic Parameters By Mstmentioning

confidence: 99%

Design and study of lipopeptide inhibitors on preventing aggregation of human islet amyloid polypeptide residues 11‐20

Mao

et al. 2017

Journal of Peptide Science

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Type 2 diabetes mellitus, a kind of conformational disease, has become an epidemic disease, which seriously endangers the quality of life and health of human beings. The deposition of human islet amyloid polypeptide (hIAPP) has been considered as one of the major pathological features of type 2 diabetes mellitus. As lipopeptides have some hydrophobic groups, which are similar to the reported aggregation inhibitors, and some lipopeptides could prevent cells from depositing of amyloid fibrils, several potential lipopeptide inhibitors have been engineered and synthesized, which have been assessed for their inhibitory effect in preventing amyloid fibrils formation of hIAPP [11][12][13][14][15][16][17][18][19][20] by using the conventional thioflavin-T fluorescence assay and new technique microscale thermophoresis (MST). The final amyloid fibrils of hIAPP [11][12][13][14][15][16][17][18][19][20] were characterized by transmission electron microscopy. Results suggested that with the increasing length of alkyl chain, the antiaggregation efficiency of lipopeptide inhibitors towards hIAPP 11-20 increased gradually.Meanwhile, the amount of arginines, which represent the head groups of lipopeptides, may also have some influence. The binding events also showed that the inhibitory efficiency of these lipopeptide inhibitors was enhanced with the increase of affinities between lipopeptides and hIAPP [11][12][13][14][15][16][17][18][19][20] , which were obtained from MST. This study demonstrated the efficiency of lipopeptides in inhibiting the aggregation of hIAPP [11][12][13][14][15][16][17][18][19][20] and proved that MST could be regarded as an appropriate and rapid method to screen potential inhibitors of hIAPP [11][12][13][14][15][16][17][18][19][20] or other amyloid proteins. This study also broadens the types of inhibitors on inhibiting amyloid formation of hIAPP.

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“…G protein binding. We next used a microscale thermophoresis technique (35) to investigate the ability of the TSHR ICD mimetics to bind recombinant purified Ga subunits. TSHR is remarkable in its ability to activate all known G protein isoforms (21)(22)(23).…”

Section: Construction Of Tshr Icd Mimeticsmentioning

confidence: 99%

Construction of Structural Mimetics of the Thyrotropin Receptor Intracellular Domain

Press

Zvagelsky

Vyazmensky

et al. 2016

Biophysical Journal

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The signaling of a G protein-coupled receptor (GPCR) is dictated by the complementary responsiveness of interacting intracellular effectors such as G proteins. Many GPCRs are known to couple to more than one G protein subtype and induce a multitude of signaling pathways, although the in vivo relevance of particular pathways is mostly unrecognized. Dissecting GPCR signaling in terms of the pathways that are activated will boost our understanding of the molecular fundamentals of hormone action. The structural determinants governing the selectivity of GPCR/G protein coupling, however, remain obscure. Here, we describe the design of soluble GPCR mimetics to study the details of the interplay between G-proteins and activators. We constructed functional mimetics of the intracellular domain of a model GPCR, the thyrotropin receptor. We based the construction on a unique scaffold, 6-Helix, an artificial protein that was derived from the elements of the trimer-of-hairpins structure of HIV gp41 and represents a bundle of six a-helices. The 6-Helix scaffold, which endowed the substituted thyrotropin receptor intracellular domain elements with spatial constraints analogous to those found in native receptors, enabled the reconstitution of a microdomain that consists of intracellular loops 2 and 3, and is capable of binding and activating Ga-(s). The 6-Helix-based mimetics could be used as a platform to study the molecular basis of GPCR/G protein recognition. Such knowledge could help investigators develop novel therapeutic strategies for GPCR-related disorders by targeting the GPCR/G protein interfaces and counteracting cellular dysfunctions via focused tuning of GPCR signaling.

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Protein-binding assays in biological liquids using microscale thermophoresis

Cited by 978 publications

References 36 publications

Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin

Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin

Design and study of lipopeptide inhibitors on preventing aggregation of human islet amyloid polypeptide residues 11‐20

Construction of Structural Mimetics of the Thyrotropin Receptor Intracellular Domain

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