Protein-binding assays in biological liquids using microscale thermophoresis.
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TLDR
The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum, which may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.Abstract:
Protein interactions inside the human body are expected to differ from the situation in vitro. This is crucial when investigating protein functions or developing new drugs. In this study, we present a sample-efficient, free-solution method, termed microscale thermophoresis, that is capable of analysing interactions of proteins or small molecules in biological liquids such as blood serum or cell lysate. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human interferon gamma and the interaction of calmodulin with calcium. The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This information about the influence of the biological matrix may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.read more
Citations
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Journal ArticleDOI
Molecular Interaction Studies Using Microscale Thermophoresis
TL;DR: This review focuses on the theoretical background of MST and gives a detailed overview on various applications to demonstrate the broad applicability.
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Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions
Susanne A. I. Seidel,Patricia M. Dijkman,Wendy A. Lea,Geert van den Bogaart,Moran Jerabek-Willemsen,Ana Lazic,Jeremiah S. Joseph,Prakash Srinivasan,Philipp Baaske,Anton Simeonov,Ilia Katritch,Fernando A. Melo,John E. Ladbury,Gideon Schreiber,Anthony Watts,Dieter Braun,Stefan Duhr +16 more
TL;DR: Flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which is demonstrated by addressing typically challenging types of binding events from various fields of life science.
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The invasin D protein from Yersinia pseudotuberculosis selectively binds the Fab region of host antibodies and affects colonization of the intestine
Pooja Sadana,Rebecca Geyer,Joern Pezoldt,Saskia Helmsing,Jochen Huehn,Michael Hust,Petra Dersch,Andrea Scrima +7 more
TL;DR: The first crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold that, apart from the canonical β-sheets, comprises various modifications of and insertions into the Ig-core structure, suggesting that InvD modulates Ig functions in the intestine and affects direct interactions with a subset of cell surface-exposed B-cell receptors.
Journal ArticleDOI
MicroScale Thermophoresis: Interaction analysis and beyond
Moran Jerabek-Willemsen,Timon André,Randy Wanner,Heide Marie Roth,Stefan Duhr,Philipp Baaske,Dennis Breitsprecher +6 more
TL;DR: This review presents recent progress and developments in MST technology and introduces alternative MST applications – such as determination of binding stoichiometries and binding modes, analysis of protein unfolding, thermodynamics and enzyme kinetics, as well as demonstrating the capability of MST to quantify high-affinity interactions with dissociation constants in the low picomolar (pM) range.
Journal ArticleDOI
Histone H3 trimethylation at lysine 36 guides m 6 A RNA modification co-transcriptionally
Huilin Huang,Huilin Huang,Hengyou Weng,Hengyou Weng,Ke-Ren Zhou,Tong Wu,Boxuan Simen Zhao,Mingli Sun,Mingli Sun,Zhenhua Chen,Xiaolan Deng,Xiaolan Deng,Xiaolan Deng,Gang Xiao,Franziska Auer,Lars Klemm,Huizhe Wu,Huizhe Wu,Huizhe Wu,Zhixiang Zuo,Zhixiang Zuo,Xi Qin,Xi Qin,Yunzhu Dong,Yile Zhou,Hanjun Qin,Shu Tao,Juan Du,Jun Liu,Zhike Lu,Hang Yin,Ana Mesquita,Celvie L. Yuan,Yueh-Chiang Hu,Wen-Ju Sun,Rui Su,Rui Su,Lei Dong,Lei Dong,Chao Shen,Chao Shen,Chenying Li,Chenying Li,Ying Qing,Ying Qing,Xi Jiang,Xiwei Wu,Miao Sun,Miao Sun,Jun-Lin Guan,Liang-Hu Qu,Minjie Wei,Markus Müschen,Gang Huang,Chuan He,Jian-Hua Yang,Jianjun Chen,Jianjun Chen +57 more
TL;DR: It is reported that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally, and it is shown that m 6A modifications are enriched in the vicinity of H3K 36me3 peaks, and are reduced globally when cellular H 3K36 me3 is depleted.
References
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Calmodulin plays a pivotal role in cellular regulation
TL;DR: The role of calcium ions (Ca2+) in cell function is beginning to be unraveled at the molecular level as a result of recent research on calcium-binding proteins and particularly on calmodulin.
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Regulation of interferon-gamma during innate and adaptive immune responses.
TL;DR: The epigenetic modifications and three-dimensional structure of the Ifng locus in naive CD4 T cells, and the modifications they undergo as these cells differentiate into effector T cells suggest a model whereby the chromatin architecture of Ifng is poised to facilitate either rapid opening or silencing during Th1 or Th2 differentiation, respectively.
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Structural Determinants of Phosphoinositide 3-kinase Inhibition by Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine
Edward H. Walker,Michael E. Pacold,Olga Perisic,Len R. Stephens,Philip T. Hawkins,Matthias P. Wymann,Roger L. Williams +6 more
TL;DR: Interestingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations.
Journal ArticleDOI
Why molecules move along a temperature gradient
Stefan Duhr,Dieter Braun +1 more
TL;DR: Using an all-optical microfluidic fluorescence method, results are presented that determine the effective charge of DNA and beads over a size range that is not accessible with electrophoresis, and support a unifying theory based on solvation entropy.