H/D Exchange- and Mass Spectrometry-Based Strategy for the Thermodynamic Analysis of Protein−Ligand Binding

2007 ◽  
Vol 79 (15) ◽  
pp. 5869-5877 ◽  
Author(s):  
Liangjie Tang ◽  
Erin D. Hopper ◽  
Yan Tong ◽  
Jack D. Sadowsky ◽  
Kimberly J. Peterson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ligand Binding ◽  
Thermodynamic Analysis

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.

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