Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Native holdup (nHU) to measure binding affinities from cell extracts.

ABSTRACT: Characterizing macromolecular interactions is essential for understanding cellular processes, yet most methods currently used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to estimate equilibrium binding constants of protein interactions directly from cell extracts. Compared to other pull-down-based assays, nHU requires less sample preparation and can be coupled to any analytical methods as readouts, such as Western blotting or mass spectrometry. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

SUBMITTER: Zambo B

PROVIDER: S-EPMC9770967 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Native holdup (nHU) to measure binding affinities from cell extracts.

Zambo Boglarka B Morlet Bastien B Negroni Luc L Trave Gilles G Gogl Gergo G

Science advances 20221221 51

Characterizing macromolecular interactions is essential for understanding cellular processes, yet most methods currently used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to estimate equilibrium binding constants of protein interactions directly from cell extracts. Compared to other pull-down-based assays, nHU requires less sample preparation and can be coupled to any analytical methods as readouts, such as Western blotting or mas ...[more]

PMID: 36542723

Similar Datasets

Native holdup (nHU) to measure binding affinities from cell extracts

Project description:Characterizing macromolecular interactions is essential for understanding cellular processes, yet nearly all methods used to detect protein interactions from cells are qualitative. Here, we introduce a native holdup (nHU) approach that requires minimal sample preparation, investigates binding mechanisms, and quantifies equilibrium binding constants of protein interactions from cell extracts. We use nHU coupled with western blotting (nHU-WB) and mass spectrometry (nHU-MS) to explore interactions of SNX27, a cargo adaptor of the retromer complex. We find good agreement between affinities measured using minimal binding fragments and those measured directly from cell extracts using nHU. This challenges the unwritten paradigm stating that biophysical parameters like binding constants cannot be accurately determined from cells or cellular extracts. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

2023-03-11 | PXD034790 | Pride

Native holdup (nHU) to measure binding affinities from cell extracts

Project description:Characterizing macromolecular interactions is essential for understanding cellular processes, yet nearly all methods used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to quantify equilibrium binding constants and explore binding mechanisms of protein interactions from cell extracts. Compared to other pulldown-based assays, nHU requires less sample preparation and can be coupled to any analytical methods, such as western blotting (nHU-WB) or mass spectrometry (nHU-MS) as readouts. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. This challenges the unwritten paradigm stating that biophysical parameters like binding constants cannot be accurately determined from cells or cellular extracts. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

2023-03-11 | PXD036024 | Pride

How to measure and evaluate binding affinities.

Project description:Quantitative measurements of biomolecule associations are central to biological understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput technologies and the projected increase in the availability of binding data, we found it especially timely to evaluate the current standards for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concentration regime were not documented, making it impossible to determine measurement reliability. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biological interpretations. Given these challenges, we provide a framework for a broad range of researchers to evaluate, teach about, perform, and clearly document high-quality equilibrium binding measurements. We apply this framework and explain underlying fundamental concepts through experimental examples with the RNA-binding protein Puf4.

| S-EPMC7452723 | biostudies-literature

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay.

Project description:Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes.

| S-EPMC4521981 | biostudies-literature

A simple method to measure CLOCK-BMAL1 DNA binding activity in tissue and cell extracts.

Project description:The proteins CLOCK and BMAL1 form a heterodimeric transcription factor essential to circadian rhythms in mammals. Daily rhythms of CLOCK-BMAL1 DNA binding activity are known to oscillate with target gene expression in vivo. Here we present a highly sensitive assay that recapitulates native CLOCK-BMAL1 DNA binding rhythms from crude tissue extracts, which we call the Clock Protein-DNA Binding Assay (CPDBA). This method can detect less than 2-fold differences in DNA binding activity, and can deliver results in two hours or less using 10 microliters (~10 micrograms) or less of crude extract, while requiring neither specialized equipment nor expensive probes. To demonstrate the sensitivity and versatility of this assay, we show that enzymatic removal of phosphate groups from proteins in tissue extracts or pharmacological inhibition of casein kinase I in cell culture increased CLOCK-BMAL1 DNA binding activity by ~1.5 to ~2 fold, as measured by the CPDBA. In addition, we show that the CPDBA can measure CLOCK-BMAL1 binding to reconstituted chromatin. The CPDBA is a sensitive, fast, efficient and versatile probe of clock function.

| S-EPMC5580408 | biostudies-literature

Nanoscale Ion Emitters in Native Mass Spectrometry for Measuring Ligand-Protein Binding Affinities.

Project description:Electrospray ionization (ESI) mass spectrometry (MS) is a crucial method for rapidly determining the interactions between small molecules and proteins with ultrahigh sensitivity. However, nonvolatile molecules and salts that are often necessary to stabilize the native structures of protein-ligand complexes can readily adduct to protein ions, broaden spectral peaks, and lower signal-to-noise ratios in native MS. ESI emitters with narrow tip diameters (∼250 nm) were used to significantly reduce the extent of adduction of salt and nonvolatile molecules to protein complexes to more accurately measure ligand-protein binding constants than by use of conventional larger-bore emitters under these conditions. As a result of decreased salt adduction, peaks corresponding to protein-ligand complexes that differ in relative molecular weight by as low as 0.06% can be readily resolved. For low-molecular-weight anion ligands formed from sodium salts, anion-bound and unbound protein ions that differ in relative mass by 0.2% were completely baseline resolved using nanoscale emitters, which was not possible under these conditions using conventional emitters. Owing to the improved spectral resolution obtained using narrow-bore emitters and an analytically derived equation, K d values were simultaneously obtained for at least six ligands to a single druggable protein target from one spectrum for the first time. This research suggests that ligand-protein binding constants can be directly and accurately measured from solutions with high concentrations of nonvolatile buffers and salts by native MS.

| S-EPMC6396573 | biostudies-literature

BANC-seq to identify genome-wide transcription factor binding affinities to native chromatin

Project description:BANC-seq (Binding Affinities to Native Chromatin by sequencing) allows determination of absolute apparent binding affinites of transcription factors to native chromatin in a genome-wide manner. In this study, we establish the method and show that chromatin and DNA sequence define binding affinites of FOXA1, SP1, YY1 and MYC/MAX complex. To relate the identified binding affinities to the actual transcriptional levels of these transcription factors in the cells used in this study, and to confirm that the nuclear isolation protocol used in the study does not lead to loss of nuclear proteins, we have generated whole proteomes including proteomics standards for absolute quantification of proteins. In addition, to validate some of the findings of the study, we have performed PAQMAN (Protein-nucleic acid affinity quantification by MAss spectrometry in nuclear extracts), as well as DNA pulldowns followed by mass spec.

2023-02-09 | PXD038502 | Pride

FoldAffinity: binding affinities from nDSF experiments.

Project description:Differential scanning fluorimetry (DSF) using the inherent fluorescence of proteins (nDSF) is a popular technique to evaluate thermal protein stability in different conditions (e.g. buffer, pH). In many cases, ligand binding increases thermal stability of a protein and often this can be detected as a clear shift in nDSF experiments. Here, we evaluate binding affinity quantification based on thermal shifts. We present four protein systems with different binding affinity ligands, ranging from nM to high μM. Our study suggests that binding affinities determined by isothermal analysis are in better agreement with those from established biophysical techniques (ITC and MST) compared to apparent Kds obtained from melting temperatures. In addition, we describe a method to optionally fit the heat capacity change upon unfolding ([Formula: see text]) during the isothermal analysis. This publication includes the release of a web server for easy and accessible application of isothermal analysis to nDSF data.

| S-EPMC8099913 | biostudies-literature

Exploring pH Dependent Host/Guest Binding Affinities.

Project description:When the electrostatic environment surrounding binding partners changes between unbound and bound states, the net uptake or release of a proton is possible by either binding partner. This process is pH-dependent in that the free energy required to uptake or release the proton varies with pH. This pH-dependence is typically not considered in conventional free energy methods where the use of fixed protonation states is the norm. In the present paper, we apply a simple two-step approach to calculate the pH-dependent binding free energy of a model cucubit[7]uril host/guest system. By use of λ-dynamics with an enhanced sampling protocol, adaptive landscape flattening, pKa shifts and reference binding free energies upon complexation were determined. This information enables the construction of pH-dependent binding profiles that accurately capture the pKa shifts and reproduce binding free energies at the different pH conditions that were observed experimentally. Our calculations illustrate a general framework for computing pH-dependent binding free energies but also point to some issues in modeling the molecular charge distributions within this series of molecules with CGenFF. However, by introducing some minor charge modifications to the CGenFF force field, we saw significant improvement in accuracy of the calculated pKa shifts.

| S-EPMC7442968 | biostudies-literature

Detecting Protein Communities in Native Cell Extracts by Machine Learning: A Structural Biologist's Perspective.

Project description:Native cell extracts hold great promise for understanding the molecular structure of ordered biological systems at high resolution. This is because higher-order biomolecular interactions, dubbed as protein communities, may be retained in their (near-)native state, in contrast to extensively purifying or artificially overexpressing the proteins of interest. The distinct machine-learning approaches are applied to discover protein-protein interactions within cell extracts, reconstruct dedicated biological networks, and report on protein community members from various organisms. Their validation is also important, e.g., by the cross-linking mass spectrometry or cell biology methods. In addition, the cell extracts are amenable to structural analysis by cryo-electron microscopy (cryo-EM), but due to their inherent complexity, sorting structural signatures of protein communities derived by cryo-EM comprises a formidable task. The application of image-processing workflows inspired by machine-learning techniques would provide improvements in distinguishing structural signatures, correlating proteomic and network data to structural signatures and subsequently reconstructed cryo-EM maps, and, ultimately, characterizing unidentified protein communities at high resolution. In this review article, we summarize recent literature in detecting protein communities from native cell extracts and identify the remaining challenges and opportunities. We argue that the progress in, and the integration of, machine learning, cryo-EM, and complementary structural proteomics approaches would provide the basis for a multi-scale molecular description of protein communities within native cell extracts.

| S-EPMC8082361 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data