Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST)

Al-Jubair, Tamim; Steffen, Jonas Hyld; Missel, Julie Winkel; Kitchen, Philip; Salman, Mootaz M.; Bill, Roslyn M.; Gourdon, Pontus; Törnroth-Horsefield, Susanna

doi:10.1016/j.xpro.2022.101316

“…There are sometimes difficulties to properly understand the thermodiffusion process in a biological context, but nevertheless the methodology is extremely useful and largely deployed [ 109 ]. Here, we essentially focused on PPIs, but the method is adaptable to other systems, such as aptamers, lipids and other types of biomolecular interactions [ 44 , 110 , 111 , 112 , 113 , 114 ]. The technology has been used for more than 15 years now and has contributed to a better understanding of many PPIs and the identification of many PPI modulators [ 115 ].…”

Section: Discussionmentioning

confidence: 99%

Microscale Thermophoresis as a Tool to Study Protein Interactions and Their Implication in Human Diseases

Magnez

¹

,

Bailly

²

,

Thuru

³

2022

IJMS

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The review highlights how protein–protein interactions (PPIs) have determining roles in most life processes and how interactions between protein partners are involved in various human diseases. The study of PPIs and binding interactions as well as their understanding, quantification and pharmacological regulation are crucial for therapeutic purposes. Diverse computational and analytical methods, combined with high-throughput screening (HTS), have been extensively used to characterize multiple types of PPIs, but these procedures are generally laborious, long and expensive. Rapid, robust and efficient alternative methods are proposed, including the use of Microscale Thermophoresis (MST), which has emerged as the technology of choice in drug discovery programs in recent years. This review summarizes selected case studies pertaining to the use of MST to detect therapeutically pertinent proteins and highlights the biological importance of binding interactions, implicated in various human diseases. The benefits and limitations of MST to study PPIs and to identify regulators are discussed.

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“…AQP2-S264E had an affinity similar to non-phosphorylated wild-type AQP2 (KD = 278 ± 49.1 nM) [111]. More recently, a protocol was developed for characterizing the binding between a human aquaporin and a soluble interaction partner using MST [112], and MST was used to measure the interaction of Zea mays aquaporin ZmPIP2;5 with the jasmonic acid analogue coronatine [113].…”

Section: Aquaporinsmentioning

confidence: 99%

Microscale Thermophoresis Analysis of Membrane Proteins

Nawaz,

Ali,

Ali

et al. 2024

CSIJ

0

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Microscale thermophoresis (MST) is an analytical technique for measuring biomolecular interactions. It is based on the physical phenomenon that particles move within temperature gradients, which is affected by their size, charge, hydration shell and conformation. The MST sample must contain a fluorescent target molecule used to observe the movement of particles, and this can be titrated with an unlabelled binding partner for quantifying the interaction. MST is highly sensitive, using relatively small amounts of sample, and it has no limitations on the size of the target biomolecule, on the affinity of the interaction or on the composition of the buffer and other sample components. This makes MST ideally suited to characterising interactions with membrane proteins, which can be studied in cell lysates, native membranes, solubilised in detergents or reconstituted in lipids. The intrinsic aromatic residues of membrane proteins have been used as the fluorophore for MST (label-free MST) or membrane proteins have been labelled with a range of fluorescent dyes or conjugated with fluorescent proteins (labelled MST). The different types of membrane proteins that have had biomolecular interactions characterised by MST include the SARS-CoV-2 spike protein, GPCRs and other receptors, sensor kinases, ion channels, aquaporins, and transport proteins.

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“…AQP2-S264E had an affinity similar to non-phosphorylated wild-type AQP2 (KD = 278 ± 49.1 nM) [67]. More recently, a protocol was developed for characterizing the binding between a human aquaporin and a soluble interaction partner using MST [68], and MST was used to measure the interaction of Zea mays aquaporin ZmPIP2;5 with the jasmonic acid analogue coronatine [69].…”

Section: Aquaporinsmentioning

confidence: 99%

Microscale Thermophoresis Analysis of Membrane Proteins

Patching

2024

Preprint

0

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Microscale thermophoresis (MST) is an analytical technique for measuring biomolecular interactions. It is based on the physical phenomenon that particles move within temperature gradients, which is affected by their size, charge, hydration shell and conformation. The MST sample must contain a fluorescent target molecule used to observe the movement of particles, and this can be titrated with an unlabelled binding partner for quantifying the interaction. MST is highly sensitive, using relatively small amounts of sample, and it has no limitations on the size of the target biomolecule, on the affinity of the interaction or on the composition of the buffer and other sample components. This makes MST ideally suited to characterising interactions with membrane proteins, which can be studied in cell lysates, native membranes, solubilised in detergents or reconstituted in lipids. The intrinsic aromatic residues of membrane proteins have been used as the fluorophore for MST (label-free MST) or membrane proteins have been labelled with a range of fluorescent dyes or conjugated with fluorescent proteins (labelled MST). The different types of membrane proteins that have had biomolecular interactions characterised by MST include the SARS-CoV-2 spike protein, GPCRs and other receptors, sensor kinases, ion channels, aquaporins, and transport proteins.

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Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST)

Cited by 3 publications

References 6 publications

Microscale Thermophoresis as a Tool to Study Protein Interactions and Their Implication in Human Diseases

Microscale Thermophoresis as a Tool to Study Protein Interactions and Their Implication in Human Diseases

Microscale Thermophoresis Analysis of Membrane Proteins

Microscale Thermophoresis Analysis of Membrane Proteins

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