Abstract
Membrane proteins are responsible for a large variety of tasks in organisms and of particular interesting as drug targets. At the same time, they are notoriously difficult to work with and require a thorough characterization before proceeding with structural studies. Here, we present a biophysical pipeline to characterize membrane proteins focusing on the optimization of stability, aggregation behavior, and homogeneity. The pipeline shown here is built on three biophysical techniques: differential scanning fluorimetry using native protein fluorescence (nano differential scanning fluorimetry), dynamic light scattering, and mass photometry. For each of these techniques, we provide detailed protocols for performing experiments and data analysis.
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References
Curnow P, Bartolo NDD, Moreton KM et al (2011) Stable folding core in the folding transition state of an α-helical integral membrane protein. Proc Natl Acad Sci U S A 108:14133–14138
Champeil P, Orlowski S, Babin S et al (2016) A robust method to screen detergents for membrane protein stabilization, revisited. Anal Biochem 511:31–35
Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14:673–681
Sonoda Y, Newstead S, Hu N-J et al (2011) Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures. Structure 19:17–25
Mancusso R, Karpowich NK, Czyzewski BK et al (2011) Simple screening method for improving membrane protein thermostability. Methods 55:324–329
Hattori M, Hibbs RE, Gouaux E (2012) A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20:1293–1299
Vergis JM, Purdy MD, Wiener MC (2010) A high-throughput differential filtration assay to screen and select detergents for membrane proteins. Anal Biochem 407:1–11
Kotov V, Bartels K, Veith K et al (2019) High-throughput stability screening for detergent-solubilized membrane proteins. Sci Rep 9:10379
Parker JL, Newstead S (2016) Membrane protein crystallisation: current trends and future perspectives. In: Advances in experimental medicine and biology. Springer International Publishing, pp 61–72
Alexandrov AI, Mileni M, Chien EYT et al (2008) Microscale fluorescent thermal stability assay for membrane proteins. Structure 16:351–359
Harris NJ, Booth PJ (2012) Folding and stability of membrane transport proteins in vitro. Biochim Biophys Acta 1818:1055–1066
Murphy RM (1997) Static and dynamic light scattering of biological macromolecules: what can we learn? Curr Opin Biotechnol 8:25–30
Raynal B, Lenormand P, Baron B et al (2014) Quality assessment and optimization of purified protein samples: why and how? Microb Cell Factories 13:180
Young G, Hundt N, Cole D et al (2018) Quantitative mass imaging of single biological macromolecules. Science 360:423–427
Hundt N (2021) Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy. Essays Biochem 65:81–91
Häußermann K, Young G, Kukura P et al (2019) Dissecting FOXP2 oligomerization and DNA binding. Angew Chem Int Ed 58:7662–7667
Soltermann F, Foley EDB, Pagnoni V et al (2020) Quantifying protein–protein interactions by molecular counting with mass photometry. Angew Chem Int Ed 59:10774–10779
Wu D, Piszczek G (2020) Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry. Anal Chem 592:113575
Olerinyova A, Sonn-Segev A, Gault J et al (2020) Mass photometry of membrane proteins. Chembiochem 7:224–236
Heermann T, Steiert F, Ramm B et al (2021) Mass-sensitive particle tracking (MSPT) to elucidate the membrane-associated MinDE reaction cycle. Nat Methods 18:1239–1246
Steiert F, Heermann T, Hundt N et al (2022) Mass-sensitive particle tracking to characterize membrane-associated macromolecule dynamics. J Vis Exp 180:e63583
Wu D, Piszczek G (2021) Standard protocol for mass photometry experiments. Eur Biophys J 50:403–409
Burastero O, Niebling S, Defelipe LA et al (2021) eSPC: an online data-analysis platform for molecular biophysics. Acta Cryst D 77:1241–1250
Kotov V, Mlynek G, Vesper O et al (2021) In-depth interrogation of protein thermal unfolding data with MoltenProt. Protein Sci 30:201–217
Bedouelle H (2016) Principles and equations for measuring and interpreting protein stability: from monomer to tetramer. Biochimie 121:29–37
Mazurenko S, Kunka A, Beerens K et al (2017) Exploration of protein unfolding by modelling calorimetry data from reheating. Sci Rep 7:16321
Koppel DE (1972) Analysis of macromolecular polydispersity in intensity correlation spectroscopy: the method of cumulants. J Chem Phys 57:4814–4820
Provencher SW (1982) A constrained regularization method for inverting data represented by linear algebraic or integral equations. Comput Phys Commun 27:213–227
Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78:1606–1619
Provencher SW (1982) CONTIN: a general purpose constrained regularization program for inverting noisy linear algebraic and integral equations. Comput Phys Commun 27:229–242
Zhao H, Schuck P (2012) Global multi-method analysis of affinities and cooperativity in complex systems of macromolecular interactions. Anal Chem 84:9513–9519
Niebling S, Veith K, Vollmer B et al (2022) Biophysical screening pipeline for Cryo-EM grid preparation of membrane proteins. Front Mol Biosci 9:882288
Noble AJ, Dandey VP, Wei H et al (2018) Routine single particle CryoEM sample and grid characterization by tomography. elife 7:e34257
Garcia-Alai MM, Heidemann J, Skruzny M et al (2018) Epsin and Sla2 form assemblies through phospholipid interfaces. Nat Commun 9:328
Lizarrondo J, Klebl DP, Niebling S et al (2021) Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis. Nat Commun 12:2889
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Niebling, S., Burastero, O., García-Alai, M. (2023). Biophysical Characterization of Membrane Proteins. In: Sousa, Â., Passarinha, L. (eds) Advanced Methods in Structural Biology. Methods in Molecular Biology, vol 2652. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3147-8_12
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DOI: https://doi.org/10.1007/978-1-0716-3147-8_12
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