scholarly journals A non-radioactive mass spectrometry based differential radial capillary action of ligand assay (DRaCALA) to assess ligand binding to proteins

2021 ◽  
Author(s):  
Annika Cimdins-Ahne ◽  
Alexey Chernobrovkin ◽  
Roman Zubarev ◽  
Ute Römling
Keyword(s):  
Mass Spectrometry ◽  
Ligand Binding ◽  
Chronic Infection ◽  
Second Messengers ◽  
Physicochemical Characteristics ◽  
Diguanylate Cyclase ◽  
Capillary Action ◽  
Ggdef Domain ◽  
Nucleotide Binding Proteins ◽  
Bacterial Domain

Binding of ligands to macromolecules changes their physicochemical characteristics. Cyclic di-GMP and other cyclic di-nucleotides are second messengers involved in motility/sessility and acute/chronic infection life style transition. Although the GGDEF domain encoding preferentially a diguanylate cyclase represents one of the most abundant bacterial domain superfamilies, the number of cyclic di-GMP receptors falls short. To facilitate screening for cyclic di-nucleotide binding proteins, we describe a non-radioactive, MALDI-TOF based modification of the widely applied differential radial capillary action of ligand assay (DRaCALA). The results of this assay suggest that YciRFec101, but not the YciRTOB1 variant of the diguanylate cyclase/phosphodiesterase YciR binds cyclic di-GMP.

scholarly journals Formation of a complex between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Shi-qi An ◽  
Ji-liang Tang
Keyword(s):  
Virulence Factors ◽  
Xanthomonas Campestris ◽  
Adaptor Protein ◽  
Physical Interaction ◽  
Diguanylate Cyclase ◽  
Domain Interaction ◽  
Ggdef Domain ◽  
Interdomain Interactions ◽  
Xanthomonas Campestris Pv Campestris ◽  
Two Hybrid

RpfG is a member of a class of wide spread bacterial two-component regulators with an HD-GYP cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), RpfG together with the sensor kinase RpfC regulates the synthesis of a range of virulence factors as a response to the cell-cell Diffusible Signaling Factor (DSF). RpfG regulates many different virulence factors by divergent pathways. Physical interaction of RpfG with two diguanylate cyclase (GGDEF) domain proteins controls motility. This is a dynamic interaction that depends upon DSF signaling and involves the conserved GYP motif in the HD-GYP domain. Here we use synthetic peptide overlay technology and yeast two-hybrid analysis in conjunction with alanine substitution mutagenesis to define a motif within the GGDEF domain proteins required for interaction. We show that regulation of motility by the GGDEF domain proteins depends upon this motif. Furthermore, we show by Y2H that both GGDEF domain proteins bind a specific PilZ domain adaptor protein, and this interacts with the pilus motor proteins PilU and PiIT. The results support a model in which DSF signaling influences motility through the interaction of proteins that affect pilus action. The motif required for HD-GYP domain interaction is conserved in a number of GGDEF domain proteins, suggesting that regulation via interdomain interactions may be of broad relevance.

scholarly journals New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain

2022 ◽  
Vol 5 (4) ◽  
pp. e202101301
Author(s):  
Ralph T Böttcher ◽  
Nico Strohmeyer ◽  
Jonas Aretz ◽  
Reinhard Fässler
Keyword(s):  
Mass Spectrometry ◽  
Ligand Binding ◽  
Phosphorylation Site ◽  
Cell Spreading ◽  
Mouse Cell ◽  
Β1 Integrin ◽  
Β1 Integrins ◽  
Integrin Ligand ◽  
Human And Mouse ◽  
Major Phosphorylation Site

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

scholarly journals Unpicking allosteric mechanisms of homo-oligomeric proteins by determining their successive ligand binding constants

2018 ◽  
Vol 373 (1749) ◽  
pp. 20170176 ◽  
Author(s):  
Ranit Gruber ◽  
Amnon Horovitz
Keyword(s):  
Mass Spectrometry ◽  
Ligand Binding ◽  
Single Molecule ◽  
Binding Constants ◽  
Molecular Machines ◽  
Native Mass Spectrometry ◽  
Oligomeric Proteins ◽  
Allosteric Mechanisms ◽  
The Relationship

Advances in native mass spectrometry and single-molecule techniques have made it possible in recent years to determine the values of successive ligand binding constants for large multi-subunit proteins. Given these values, it is possible to distinguish between different allosteric mechanisms and, thus, obtain insights into how various bio-molecular machines work. Here, we describe for ring-shaped homo-oligomers, in particular, how the relationship between the values of successive ligand binding constants is diagnostic for concerted, sequential and probabilistic allosteric mechanisms. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.

scholarly journals Probing ligand and cation binding sites in G-quadruplex nucleic acids by mass spectrometry and electron photodetachment dissociation sequencing

The Analyst ◽  
2019 ◽  
Vol 144 (11) ◽  
pp. 3518-3524 ◽  
Author(s):  
Dababrata Paul ◽  
Adrien Marchand ◽  
Daniela Verga ◽  
Marie-Paule Teulade-Fichou ◽  
Sophie Bombard ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Ligand Binding ◽  
Binding Sites ◽  
Cation Binding ◽  
Tandem Mass ◽  
Top Down ◽  
G Quadruplex ◽  
Ligand Binding Sites ◽  
Electron Photodetachment

Tandem mass spectrometry: native top-down sequencing by electron photodetachment dissociation (EPD) reveals ligand binding sites on DNA G-quadruplexes.

scholarly journals Steroid and Protein Ligand Binding to Cytochrome P450 46A1 as Assessed by Hydrogen−Deuterium Exchange and Mass Spectrometry

Biochemistry ◽  
2009 ◽  
Vol 48 (19) ◽  
pp. 4150-4158 ◽  
Author(s):  
Wei-Li Liao ◽  
Nathan G. Dodder ◽  
Natalia Mast ◽  
Irina A. Pikuleva ◽  
Illarion V. Turko
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Ligand Binding ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Hydrogen Deuterium

A General Mass Spectrometry-Based Assay for the Quantitation of Protein−Ligand Binding Interactions in Solution

2002 ◽  
Vol 124 (35) ◽  
pp. 10256-10257 ◽  
Author(s):  
Kendall D. Powell ◽  
Sina Ghaemmaghami ◽  
Michael Z. Wang ◽  
Liyuan Ma ◽  
Terrance G. Oas ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ligand Binding ◽  
Binding Interactions ◽  
General Mass
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