Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains.

ABSTRACT: We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution.

SUBMITTER: Isaksson L

PROVIDER: S-EPMC3647075 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains.

Isaksson Linnéa L Mayzel Maxim M Saline Maria M Pedersen Anders A Rosenlöw Joakim J Brutscher Bernhard B Karlsson B Göran BG Orekhov Vladislav Y VY

PloS one 20130507 5

We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution. ...[more]

PMID: 23667548

Similar Datasets

An assignment of intrinsically disordered regions of proteins based on NMR structures.

Project description:Intrinsically disordered proteins (IDPs) do not adopt stable three-dimensional structures in physiological conditions, yet these proteins play crucial roles in biological phenomena. In most cases, intrinsic disorder manifests itself in segments or domains of an IDP, called intrinsically disordered regions (IDRs), but fully disordered IDPs also exist. Although IDRs can be detected as missing residues in protein structures determined by X-ray crystallography, no protocol has been developed to identify IDRs from structures obtained by Nuclear Magnetic Resonance (NMR). Here, we propose a computational method to assign IDRs based on NMR structures. We compared missing residues of X-ray structures with residue-wise deviations of NMR structures for identical proteins, and derived a threshold deviation that gives the best correlation of ordered and disordered regions of both structures. The obtained threshold of 3.2Å was applied to proteins whose structures were only determined by NMR, and the resulting IDRs were analyzed and compared to those of X-ray structures with no NMR counterpart in terms of sequence length, IDR fraction, protein function, cellular location, and amino acid composition, all of which suggest distinct characteristics. The structural knowledge of IDPs is still inadequate compared with that of structured proteins. Our method can collect and utilize IDRs from structures determined by NMR, potentially enhancing the understanding of IDPs.

| S-EPMC3529935 | biostudies-literature

In-Cell NMR Spectroscopy of Intrinsically Disordered Proteins.

Project description:This review summarizes the results of in-cell Nuclear Magnetic Resonance, NMR, spectroscopic investigations of the eukaryotic and prokaryotic intrinsically disordered proteins, IDPs: α-synuclein, prokaryotic ubiquitin-like protein, Pup, tubulin-related neuronal protein, Tau, phenylalanyl-glycyl-repeat-rich nucleoporins, FG Nups, and the negative regulator of flagellin synthesis, FlgM. The results show that the cellular behavior of IDPs may differ significantly from that observed in the test tube.

| S-EPMC7004275 | biostudies-literature

Intrinsically disordered proteins: modes of binding with emphasis on disordered domains.

Project description:Our notions of protein function have long been determined by the protein structure-function paradigm. However, the idea that protein function is dictated by a prerequisite complementarity of shapes at the binding interface is becoming increasingly challenged. Interactions involving intrinsically disordered proteins (IDPs) have indicated a significant degree of disorder present in the bound state, ranging from static disorder to complete disorder, termed 'random fuzziness'. This review assesses the anatomy of an IDP and relates how its intrinsic properties permit promiscuity and allow for the various modes of interaction. Furthermore, a mechanistic overview of the types of disordered domains is detailed, while also relating to a recent example and the kinetic and thermodynamic principles governing its formation.

| S-EPMC8492171 | biostudies-literature

Amino acid recognition for automatic resonance assignment of intrinsically disordered proteins.

Project description:Resonance assignment is a prerequisite for almost any NMR-based study of proteins. It can be very challenging in some cases, however, due to the nature of the protein under investigation. This is the case with intrinsically disordered proteins, for example, whose NMR spectra suffer from low chemical shifts dispersion and generally low resolution. For these systems, sequence specific assignment is highly time-consuming, so the prospect of using automatic strategies for their assignment is very attractive. In this article we present a new version of the automatic assignment program TSAR dedicated to intrinsically disordered proteins. In particular, we demonstrate how the automatic procedure can be improved by incorporating methods for amino acid recognition and information on chemical shifts in selected amino acids. The approach was tested in silico on 16 disordered proteins and experimentally on α-synuclein, with remarkably good results.

| S-EPMC4824835 | biostudies-literature

NMR contributions to structural dynamics studies of intrinsically disordered proteins.

Project description:Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity. Given their inherent structural flexibility X-ray crystallography is not applicable to study these proteins. In contrast, NMR spectroscopy offers unique opportunities for structural and dynamic studies of IDPs. The past two decades have witnessed significant development of NMR spectroscopy that couples advances in spin physics and chemistry with a broad range of applications. This article will summarize key advances in basic physical-chemistry and NMR methodology, outline their limitations and envision future R&D directions.

| S-EPMC3985426 | biostudies-literature

Conformational ensembles explain NMR spectra of frozen intrinsically disordered proteins.

Project description:Protein regions which are intrinsically disordered, exist as an ensemble of rapidly interconverting structures. Cooling proteins to cryogenic temperatures for dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR studies suspends most of the motions, resulting in peaks that are broad but not featureless. To demonstrate that detailed conformational restraints can be retrieved from the peak shapes of frozen proteins alone, we developed and used a simulation framework to assign peak features to conformers in the ensemble. We validated our simulations by comparing them to spectra of α-synuclein acquired under different experimental conditions. Our assignments of peaks to discrete dihedral angle populations suggest that structural constraints are attainable under cryogenic conditions. The ability to infer ensemble populations from peak shapes has important implications for DNP MAS NMR studies of proteins with regions of disorder in living cells because chemical shifts are the most accessible measured parameter.

| S-EPMC10108432 | biostudies-literature

αα-Hub domains and intrinsically disordered proteins: A decisive combo.

Project description:Hub proteins are central nodes in protein-protein interaction networks with critical importance to all living organisms. Recently, a new group of folded hub domains, the αα-hubs, was defined based on a shared αα-hairpin supersecondary structural foundation. The members PAH, RST, TAFH, NCBD, and HHD are found in large proteins such as Sin3, RCD1, TAF4, CBP, and harmonin, which organize disordered transcriptional regulators and membrane scaffolds in interactomes of importance to human diseases and plant quality. In this review, studies of structures, functions, and complexes across the αα-hubs are described and compared to provide a unified description of the group. This analysis expands the associated molecular concepts of "one domain-one binding site", motif-based ligand binding, and coupled folding and binding of intrinsically disordered ligands to additional concepts of importance to signal fidelity. These include context, motif reversibility, multivalency, complex heterogeneity, synergistic αα-hub:ligand folding, accessory binding sites, and supramodules. We propose that these multifaceted protein-protein interaction properties are made possible by the characteristics of the αα-hub fold, including supersite properties, dynamics, variable topologies, accessory helices, and malleability and abetted by adaptability of the disordered ligands. Critically, these features provide additional filters for specificity. With the presentations of new concepts, this review opens for new research questions addressing properties across the group, which are driven from concepts discovered in studies of the individual members. Combined, the members of the αα-hubs are ideal models for deconvoluting signal fidelity maintained by folded hubs and their interactions with intrinsically disordered ligands.

| S-EPMC7948954 | biostudies-literature

Five and four dimensional experiments for robust backbone resonance assignment of large intrinsically disordered proteins: application to Tau3x protein.

Project description:New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.

| S-EPMC4983291 | biostudies-other

Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments.

Project description:A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high dimensionality and long evolution times, allowed by non-uniform sampling in the indirect dimensions. Random distribution of the data points and Sparse Multidimensional Fourier Transform processing were used. Successful application of the assignment procedure to a particularly difficult protein, δ subunit of RNA polymerase from Bacillus subtilis, is shown to prove the efficiency of the strategy. The studied protein contains a disordered C-terminal region of 81 amino acids with a highly repetitive sequence. While the conventional assignment methods completely failed due to a very small differences in chemical shifts, the presented strategy provided a complete backbone and side-chain assignment.

| S-EPMC2966349 | biostudies-literature

Tyrosine phosphorylation within the intrinsically disordered cytosolic domains of the B-cell receptor: an NMR-based structural analysis.

Project description:Intrinsically disordered proteins are found extensively in cell signaling pathways where they often are targets of posttranslational modifications e.g. phosphorylation. Such modifications can sometimes induce or disrupt secondary structure elements present in the modified protein. CD79a and CD79b are membrane-spanning, signal-transducing components of the B-cell receptor. The cytosolic domains of these proteins are intrinsically disordered and each has an immunoreceptor tyrosine-based activation motif (ITAM). When an antigen binds to the receptor, conserved tyrosines located in the ITAMs are phosphorylated which initiate further downstream signaling. Here we use NMR spectroscopy to examine the secondary structure propensity of the cytosolic domains of CD79a and CD79b in vitro before and after phosphorylation. The phosphorylation patterns are identified through analysis of changes of backbone chemical shifts found for the affected tyrosines and neighboring residues. The number of the phosphorylated sites is confirmed by mass spectrometry. The secondary structure propensities are calculated using the method of intrinsic referencing, where the reference random coil chemical shifts are measured for the same protein under denaturing conditions. Our analysis revealed that CD79a and CD79b both have an overall propensity for α-helical structure that is greatest in the C-terminal region of the ITAM. Phosphorylation of CD79a caused a decrease in helical propensity in the C-terminal ITAM region. For CD79b, the opposite was observed and phosphorylation resulted in an increase of helical propensity in the C-terminal part.

| S-EPMC4000212 | 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