NMR Spectroscopy in the Analysis of Protein-Protein Interactions

Gell, David A.; Kwan, Ann H.; Mackay, J.P.

doi:10.1007/978-3-319-28275-6_121-1

Cited by 3 publications

(2 citation statements)

References 134 publications

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“…Changes in the two-dimensional (2D) heteronuclear single-quantum coherence spectra are then used to calculate the binding affinity and binding site of the protein complex. Several other NMR-based methods have also been developed to study membrane dynamic proteinprotein interactions, such as solvent paramagnetic relaxation enhancement, residual dipolar coupling, and nuclear Overhauser effect (Vinogradova and Qin, 2012;Gell et al, 2017;Larsen et al, 2018).…”

Section: Nuclear Magnetic Resonance Spectroscopymentioning

confidence: 99%

Current Methods for Detecting Cell Membrane Transient Interactions

2020

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Short-lived cell membrane complexes play a key role in regulating cell signaling and communication. Many of these complexes are formed based on low-affinity and transient interactions among various lipids and proteins. New techniques have emerged to study these previously overlooked membrane transient interactions. Exciting functions of these transient interactions have been discovered in cellular events such as immune signaling, host–pathogen interactions, and diseases such as cancer. In this review, we have summarized current experimental methods that allow us to detect and analyze short-lived cell membrane protein–protein, lipid–protein, and lipid–lipid interactions. These methods can provide useful information about the strengths, kinetics, and/or spatial patterns of membrane transient interactions. However, each method also has its own limitations. We hope this review can be used as a guideline to help the audience to choose proper approaches for studying membrane transient interactions in different membrane trafficking and cell signaling events.

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Section: Nuclear Magnetic Resonance Spectroscopymentioning

confidence: 99%

Current Methods for Detecting Cell Membrane Transient Interactions

2020

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“…Selective isotope-labelling presents an elegant way for improving the spectral resolution (Verardi et al 2012 ) with site-selective 13 C and 2 H labelling delivering greatly simplified protein NMR spectra, which benefit the analysis of large proteins (Takeuchi et al 2007 ; Kainosho and Güntert 2009 ; Takeda et al 2010 ). As a drawback, the cost of sample preparation with isotope-labelled amino acids can be high (Gell et al 2017 ). The present work presents a new strategy for selective 13 C-labelling of aromatic amino acids that uses inexpensive 13 C-labelled precursors to direct 13 C labels into specific positions of the aromatic side chains of phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr) in a cell-free protein synthesis (CFPS) system.…”

Section: Introductionmentioning

confidence: 99%

Improved spectral resolution of [13C,1H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive 13C-labelled precursors

2023

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Cell-free protein synthesis using eCells allows production of amino acids from inexpensive 13C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of 13C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [13C,1H]-HSQC cross-peaks free of one-bond 13C–13C couplings. Selective 13C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.

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Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy

Marcano-García,

Zaza,

Dalby

et al. 2024

Nano Lett.

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Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein−protein association (K a ) and dissociation (K d ) constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method's accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.

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NMR Spectroscopy in the Analysis of Protein-Protein Interactions

Cited by 3 publications

References 134 publications

Current Methods for Detecting Cell Membrane Transient Interactions

Current Methods for Detecting Cell Membrane Transient Interactions

Improved spectral resolution of [13C,1H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive 13C-labelled precursors

Quantitative Analysis of Protein–Protein Equilibrium Constants in Cellular Environments Using Single-Molecule Localization Microscopy

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