Assessing the Stability of Membrane Proteins to Detect Ligand Binding Using Differential Static Light Scattering

Ghanei, Hamed; Khutoreskaya, G.; Dobrovetsky, E.; Edwards, A.M.; Privé, Gilbert G.; Vedadi, Masoud

doi:10.1177/1087057109357117

Cited by 27 publications

(25 citation statements)

References 35 publications

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“…Thermostability assays using fluorescent probes to monitor unfolding of soluble proteins are now routine, but similar approaches are tricky with membrane proteins because of their hydrophobic nature 20 . Instead, we turned to a recently-developed differential static light scattering technology in 384-well format that has been used predominantly to detect changes in protein stability upon ligand binding 21 .…”

Section: Resultsmentioning

confidence: 99%

Architectural and thermodynamic principles underlying intramembrane protease function

Baker¹,

Urban²

2012

Nat Chem Biol

127

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Intramembrane proteases hydrolyze peptide bonds within the membrane as a signaling paradigm universal to all life forms and with implications in disease. Deciphering the architectural strategies supporting intramembrane proteolysis is an essential but unattained goal. We integrated a new, quantitative and high-throughput thermal light-scattering technology, reversible equilibrium un/refolding, and quantitative protease assays to interrogate rhomboid architecture with 151 purified variants. Rhomboid proteases maintain low intrinsic thermodynamic stability (ΔG=2.1-4.5kcal/mol) resulting from a multitude of generally-weak transmembrane packing interactions, making them highly-responsive to their environment. Stability is consolidated by two buried glycines and several packing leucines, with a few multifaceted hydrogen bonds strategically-deployed to two peripheral regions. Opposite these regions lie transmembrane segment 5 and connected loops that are notably exempt of structural responsibility, suggesting intramembrane proteolysis involves considerable but localized protein dynamics. Our analyses provide a comprehensive ‘heat map’ of the physio-chemical anatomy underlying membrane-immersed enzyme function at unprecedented resolution.

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Section: Resultsmentioning

confidence: 99%

Architectural and thermodynamic principles underlying intramembrane protease function

Baker¹,

Urban²

2012

Nat Chem Biol

127

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“…The direct interaction of these compounds with hAQP1 was further tested by monitoring the aggregation temperature of the protein. The thermal shift relative to free protein (ΔT agg ) can be determined using light scattering [41]. We found that NSC657298 reduced T agg by 4.1°C, while NSC670226 caused a 1.5°C reduction (Fig.…”

Section: Binding Studies Of Two Selected Hit Compounds To Haqp1mentioning

confidence: 96%

“…Temperature-dependent protein aggregation was measured by using static light scattering (Stargazer-384, Harbinger Biotech) as previously described [41] with modifications. Briefly, purified hAQP1 in PBS pH 7.4 with 1% OG was diluted to 0.2 mg/mL, incubated with 30 μM of test compound, and aliquoted to a final volume of 45 μL in triplicates in a clear-bottom 384-well plate (Nunc).…”

Section: Thermal Stability Studies By Differential Static Light Scattmentioning

confidence: 99%

A generic high-throughput assay to detect aquaporin functional mutants: Potential application to discovery of aquaporin inhibitors

Yeo

Soon

et al. 2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…In addition, the assay volume can be miniaturized to 5 to 10 µL using low protein concentrations (0.025-0.1 mg/mL), being an attractive alternative for optical light-scattering methods. 8,13 The assay robustness is reflected by highly reproducible apparent T m values with standard deviations of 0.1 to 0.3 °C. This setup enables the reliable identification of potential ligands stabilizing the target of interest >1 °C.…”

Section: Thiol-reactive Bodipy Fl-l-cystine: a Versatile Dye To Monitmentioning

confidence: 99%

“…13 This label-free technique measures the intensity increase of scattered light during the thermal unfolding and aggregation of proteins, enabling the determination of an aggregation temperature (T agg ). In many cases, T agg correlates very well with the corresponding T m being measured by DSF.…”

Section: Good Correlation Of Cxcr2 T M and T Agg Values Determined Inmentioning

confidence: 99%

An Alternative Thiol-Reactive Dye to Analyze Ligand Interactions with the Chemokine Receptor CXCR2 Using a New Thermal Shift Assay Format

et al. 2016

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Integral membrane proteins (IMPs) play an important role in many cellular events and are involved in numerous pathological processes. Therefore, understanding the structure and function of IMPs is a crucial prerequisite to enable successful targeting of these proteins with low molecular weight (LMW) ligands early on in the discovery process. To optimize IMP purification/crystallization and to identify/characterize LMW ligand-target interactions, robust, reliable, high-throughput, and sensitive biophysical methods are needed. Here, we describe a differential scanning fluorimetry (DSF) screening method using the thiol-reactive BODIPY FL-cystine dye to monitor thermal unfolding of the G-protein-coupled receptor (GPCR), CXCR2. To validate this method, the seven-transmembrane protein CXCR2 was analyzed with a set of well-characterized antagonists. This study showed that the new DSF assay assessed reliably the stability of CXCR2 in a 384-well format. The analysis of 14 ligands with a potency range over 4 log units demonstrated the detection/characterization of LMW ligands binding to the membrane protein target. Furthermore, DSF results cross-validated with the label-free differential static light scattering (DSLS) thermal denaturation method. These results underline the potential of the BODIPY assay format as a general tool to investigate membrane proteins and their interaction partners.

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Assessing the Stability of Membrane Proteins to Detect Ligand Binding Using Differential Static Light Scattering

Cited by 27 publications

References 35 publications

Architectural and thermodynamic principles underlying intramembrane protease function

Architectural and thermodynamic principles underlying intramembrane protease function

A generic high-throughput assay to detect aquaporin functional mutants: Potential application to discovery of aquaporin inhibitors

An Alternative Thiol-Reactive Dye to Analyze Ligand Interactions with the Chemokine Receptor CXCR2 Using a New Thermal Shift Assay Format

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