Abstract
PDZ domains are small globular domains involved in protein–protein interactions. They participate in a wide range of critical cellular processes. These domains, very abundant in the human proteome, are widely studied by high-throughput interactomics approaches and by biophysical and structural methods. However, the quality of the results is strongly related to the optimal folding and solubility of the domains. We provide here a detailed description of protocols for a strict quality assessment of the PDZ constructs. We describe appropriate experimental approaches that have been selected to overcome the small size of such domains to check the purity, identity, homogeneity, stability, and folding of samples.
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Weatheritt RJ, Luck K, Petsalaki E, Davey NE, Gibson TJ (2012) The identification of short linear motif-mediated interfaces within the human interactome. Bioinformatics 28:976–982. https://doi.org/10.1093/bioinformatics/bts072
Caillet-Saguy C, Maisonneuve P, Delhommel F, Terrien E, Babault N, Lafon M, Cordier F, Wolff N (2015) Strategies to interfere with PDZ-mediated interactions in neurons: what we can learn from the rabies virus. Prog Biophys Mol Biol 119:53–59. https://doi.org/10.1016/j.pbiomolbio.2015.02.007
Luck K, Charbonnier S, Travé G (2012) The emerging contribution of sequence context to the specificity of protein interactions mediated by PDZ domains. FEBS Lett 586:2648–2661. https://doi.org/10.1016/j.febslet.2012.03.056
Ye F, Zhang M (2013) Structures and target recognition modes of PDZ domains: recurring themes and emerging pictures. Biochem J 455:1–14. https://doi.org/10.1042/BJ20130783
Wang CK, Pan L, Chen J, Zhang M (2010) Extensions of PDZ domains as important structural and functional elements. Protein Cell 1:737–751. https://doi.org/10.1007/s13238-010-0099-6
Delhommel F, Cordier F, Bardiaux B, Bouvier G, Colcombet-Cazenave B, Brier S, Raynal B, Nouaille S, Bahloul A, Chamot-Rooke J, Nilges M, Petit C, Wolff N (2017) Structural characterization of whirlin reveals an unexpected and dynamic supramodule conformation of its PDZ tandem. Structure 25:1645–1656.e5. https://doi.org/10.1016/j.str.2017.08.013
Long J, Wei Z, Feng W, Yu C, Zhao Y, Zhang M (2008) Supramodular nature of GRIP1 revealed by the structure of its PDZ12 tandem in complex with the carboxyl tail of Fras1. J Mol Biol 375:1457–1468. https://doi.org/10.1016/j.jmb.2007.11.088
Delhommel F, Chaffotte A, Terrien E, Raynal B, Buc H, Delepierre M, Cordier F, Wolff N (2015) Deciphering the unconventional peptide binding to the PDZ domain of MAST2. Biochem J 469:159–168. https://doi.org/10.1042/BJ20141198
Wu J, Yang Y, Zhang J, Ji P, Du W, Jiang P, Xie D, Huang H, Wu M, Zhang G, Wu J, Shi Y (2007) Domain-swapped dimerization of the second PDZ domain of ZO2 may provide a structural basis for the polymerization of claudins. J Biol Chem 282:35988–35999. https://doi.org/10.1074/jbc.M703826200
Chang BH, Gujral TS, Karp ES, BuKhalid R, Grantcharova VP, MacBeath G (2011) A systematic family-wide investigation reveals that ∼30% of mammalian PDZ domains engage in PDZ-PDZ interactions. Chem Biol 18:1143–1152. https://doi.org/10.1016/j.chembiol.2011.06.013
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins M, Appel RD, Bairoch AM (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, pp 571–607
Noble JE (2014) Quantification of protein concentration using UV absorbance and Coomassie dyes. Methods Enzymol 536:17–26. https://doi.org/10.1016/B978-0-12-420070-8.00002-7
Glasel JA (1995) Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. BioTechniques 18:62–63
Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423. https://doi.org/10.1002/pro.5560041120
Karas M, Hillenkamp F (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 60:2299–2301. https://doi.org/10.1021/ac00171a028
Suckau D, Resemann A (2003) T3-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry. Anal Chem 75:5817–5824. https://doi.org/10.1021/ac034362b
Philo JS (2006) Is any measurement method optimal for all aggregate sizes and types? AAPS J 8:E564–E571. https://doi.org/10.1208/aapsj080365
Nobbmann U, Connah M, Fish B, Varley P, Gee C, Mulot S, Chen J, Zhou L, Lu Y, Shen F, Yi J, Harding SE (2007) Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol Genet Eng Rev 24:117–128
Raynal B, Lenormand P, Baron B, Hoos S, England P (2010) Quality assessment and optimization of purified protein samples: why and how? Microb Cell Factories 13. https://doi.org/10.1186/s12934-014-0180-6
Fekete S, Beck A, Veuthey J-L, Guillarme D (2014) Theory and practice of size exclusion chromatography for the analysis of protein aggregates. J Pharm Biomed Anal 101:161–173. https://doi.org/10.1016/j.jpba.2014.04.011
Sahin E, Roberts CJ (2012) Size-exclusion chromatography with multi-angle light scattering for elucidating protein aggregation mechanisms. Methods Mol Biol 899:403–423. https://doi.org/10.1007/978-1-61779-921-1_25
Daviter T, Chmel N, Rodger A (2013) Circular and linear dichroism spectroscopy for the study of protein-ligand interactions. Methods Mol Biol 1008:211–241
Schuck P (2003) On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal Biochem 320:104–124. https://doi.org/10.1016/S0003-2697(03)00289-6
Demeler B (2010) Methods for the design and analysis of sedimentation velocity and sedimentation equilibrium experiments with proteins. Curr Protoc Protein Sci:1–24. https://doi.org/10.1002/0471140864.ps0713s60
Balbo A, Zhao H, Brown PH, Schuck P (2009) Assembly, loading, and alignment of an analytical ultracentrifuge sample cell. J Vis Exp:e1530. https://doi.org/10.3791/1530
Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78:1606–1619. https://doi.org/10.1016/S0006-3495(00)76713-0
Micsonai A, Wien F, Bulyáki É, Kun J, Moussong É, Lee Y-H, Goto Y, Réfrégiers M, Kardos J (2018) BeStSel: a web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra. Nucleic Acids Res 46:W315–W322. https://doi.org/10.1093/nar/gky497
Medrano G, Dolan MC, Condori J, Radin DN, Cramer CL (2012) Quality assessment of recombinant proteins produced in plants. Methods Mol Biol 824:535–564. https://doi.org/10.1007/978-1-61779-433-9_29
Structural Genomics Consortium, China Structural Genomics Consortium, Northeast Structural Genomics Consortium, Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, Dhe-Paganon S, Park H, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim S-H, Rao Z, Shi Y, Terwilliger TC, Kim C-Y, Hung L-W, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schütz A, Heinemann U, Yokoyama S, Büssow K, Gunsalus KC (2008) Protein production and purification. Nat Methods 5:135–146. https://doi.org/10.1038/nmeth.f.202
Dupeux F, Röwer M, Seroul G, Blot D, Márquez JA (2011) A thermal stability assay can help to estimate the crystallization likelihood of biological samples. Acta Crystallogr D Biol Crystallogr 67:915–919. https://doi.org/10.1107/S0907444911036225
Bruce D, Cardew E, Freitag-Pohl S, Pohl E (2019) How to stabilize protein: stability screens for thermal shift assays and nano differential scanning fluorimetry in the virus-X project. J Vis Exp. https://doi.org/10.3791/58666
Boivin S, Kozak S, Meijers R (2013) Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif 91:192–206. https://doi.org/10.1016/j.pep.2013.08.002
Genera M, Samson D, Raynal B, Haouz A, Baron B, Simenel C, Guerois R, Wolff N, Caillet-Saguy C (2019) Structural and functional characterization of the PDZ domain of the human phosphatase PTPN3 and its interaction with the human papillomavirus E6 oncoprotein. Sci Rep 9:7438. https://doi.org/10.1038/s41598-019-43932-x
Monsellier E, Bedouelle H (2005) Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength. Protein Eng Des Sel 18:445–456. https://doi.org/10.1093/protein/gzi046
Terrien E, Chaffotte A, Lafage M, Khan Z, Préhaud C, Cordier F, Simenel C, Delepierre M, Buc H, Lafon M, Wolff N (2012) Interference with the PTEN-MAST2 interaction by a viral protein leads to cellular relocalization of PTEN. Sci Signal 5:ra58. https://doi.org/10.1126/scisignal.2002941
Rath A, Glibowicka M, Nadeau VG, Chen G, Deber CM (2009) Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proc Natl Acad Sci U S A 106:1760–1765. https://doi.org/10.1073/pnas.0813167106
Shirai A, Matsuyama A, Yashiroda Y, Hashimoto A, Kawamura Y, Arai R, Komatsu Y, Horinouchi S, Yoshida M (2008) Global analysis of gel mobility of proteins and its use in target identification. J Biol Chem 283:10745–10752. https://doi.org/10.1074/jbc.M709211200
Duhoo Y, Girault V, Turchetto J, Ramond L, Durbesson F, Fourquet P, Nominé Y, Cardoso V, Sequeira AF, Brás JLA, Fontes CMGA, Travé G, Wolff N, Vincentelli R (2019) High-throughput production of a new library of human single and tandem PDZ domains allows quantitative PDZ-peptide interaction screening through high-throughput holdup assay. Methods Mol Biol 2025:439–476. https://doi.org/10.1007/978-1-4939-9624-7_21
Ivarsson Y, Wawrzyniak AM, Kashyap R, Polanowska J, Betzi S, Lembo F, Vermeiren E, Chiheb D, Lenfant N, Morelli X, Borg J-P, Reboul J, Zimmermann P (2013) Prevalence, specificity and determinants of lipid-interacting PDZ domains from an in-cell screen and in vitro binding experiments. PLoS One 8:e54581. https://doi.org/10.1371/journal.pone.0054581
Wawrzyniak AM, Kashyap R, Zimmermann P (2013) Phosphoinositides and PDZ domain scaffolds. Adv Exp Med Biol 991:41–57. https://doi.org/10.1007/978-94-007-6331-9_4
Layne E (1957) Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol 3:447–454
Lees JG, Miles AJ, Wien F, Wallace BA (2006) A reference database for circular dichroism spectroscopy covering fold and secondary structure space. Bioinformatics 22:1955–1962. https://doi.org/10.1093/bioinformatics/btl327
Page R, Peti W, Wilson IA, Stevens RC, Wüthrich K (2005) NMR screening and crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a structural genomics pipeline. Proc Natl Acad Sci U S A 102:1901–1905. https://doi.org/10.1073/pnas.0408490102
Bodenhausen G, Ruben DJ (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189. https://doi.org/10.1016/0009-2614(80)80041-8
Kwan AH, Mobli M, Gooley PR, King GF, Mackay JP (2011) Macromolecular NMR spectroscopy for the non-spectroscopist. FEBS J 278:687–703. https://doi.org/10.1111/j.1742-4658.2011.08004.x
Acknowledgments
The authors acknowledge Florence Cordier for technical support in NMR. The authors thank the molecular biophysics facility at Institut Pasteur for providing cutting-edge instruments.
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Caillet-Saguy, C., Brûlé, S., Wolff, N., Raynal, B. (2021). PDZ Sample Quality Assessment by Biochemical and Biophysical Characterizations. In: Borg, JP. (eds) PDZ Mediated Interactions. Methods in Molecular Biology, vol 2256. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1166-1_6
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