Abstract
It is important to understand the effect of crowding conditions on the native structure and functional state of enzymes. Equilibrium denaturation studies of Clarius gariepinus GST (CgGST) by guanidine hydrochloride (GdHCl) under dilute conditions and in separate solutions of 0–100 g dm−3 Ficoll 70, polyethylene glycol 6000 (PEG 6000) and equal w/v mixtures of the two polymers at 25 °C and pH 7.4 were studied fluorometrically. The data were analyzed based on a two-state model assuming the native protein dimer separates into two monomers and then unfolds. The standard free energy of unfolding (ΔG°UN) increases with increasing concentration of each crowding agent in a manner suggesting that high concentrations of PEG 6000 and Ficoll 70 favour the native CgGST relative to the unfolded form. Ficoll 70 stabilizes the native CgGST better than PEG 6000 at low w/v concentration. A mixture of equal g/cm3 concentrations of both crowding agents, however, stabilizes the native form more effectively than either Ficoll 70 or PEG 6000 at equivalent w/v total concentration and is less sensitive to GdHCl. This is in strong agreement with the results of refolding studies, and suggests that a mixture of molecular crowders of widely different molecular weights might show enhanced excluded volume effects compared to a single crowder. Thus, mixed crowding agents more effectively protect the enzyme against denaturation and assist in renaturation better than a single crowder. This suggests a heterogeneous solution of crowders, as will be found within cells, enhances the beneficial effect of crowding on the folded protein stability.
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References
Abdalla A, Hamed R (2006) Multiple unfolding of glutathione transferase from Physa acuta (Gastropada: Physidae). Biochem Biophys Res Commun 340:625–632
Arca P, Hardisson C, Suarez J (1997) Purification of a glutathione S-transferase that mediatesfosfomycin resistance in bacteria. Antimicrob Agents Chemother 34:844–848
Arduini A, Strambini G, Illio CD, Aceto A, Storto S, Federici G (1989) Tryptophan environment in glutathione transferase of human placenta from temperature dependent phosphorescence studies. Biochim Biophys Acta 999:203–207
Biswas S, Kundu J, Mukherjee SK, Chowdhury PK (2018) Mixed macromolecular crowding: a protein and solvent perspective. ACS Omega 3:4316–4330
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chiti F, Dobson CM (2017) Protein misfolding, amyloid formation and human disease: a summary of progress over the last decade. Ann Rev Biochem 86:27–68
Donovan JW (1969) Changes in ultraviolet absorption produced by alteration of protein conformation. J Biol Chem 244:1961–1967
Eftink MR (1994) The use of fluorescence methods to monitor unfolding transitions. Biophys J 66:482–501
Ellis RJ (2006) Molecular chaperones: assisting assembly in addition to folding. Trends Biochem Sci 31:395–401
Fernandez C, Fodeke AA and Minton AP (2019) Quantitative characterization of the concentration-dependent interaction between molecules of dextran 70 in aqueous solution: measurement and analysis in context of thermodynamic and compressible sphere models. Biopolymers 110(5):1–6 e23284. https://doi.org/10.1002/bip.23284
Fodeke AA and Minton AP (2010) Quantitative characterization of polymer–polymer, protein–protein, and polymer–protein interaction via tracer sedimentation equilibrium. J Phys Chem B 114:10876–10880. https://pubs.acs.org/doi/abs/10.1021/jp104342f
Fodeke AA, Minton AP (2011) Quantitative characterization of temperature-independent and temperature-dependent protein-protein interactions in highly nonideal solutions. J Phys Chem B 115(38):11261–11268
Fodeke AA (2019) Quantitative characterization of temperature-independent polymer–polymer interaction and temperature-dependent protein–protein and protein–polymer interactions in concentrated. Polymer solutions. Eur Biophys J https://link.springer.com/10.1007/s00249-019-01345-8
Habig WH, Pabst MJ, Jacoby WD (1974) Glutathione-S-transferases the first step in mercapturic acid formation. J Biol Chem 249:7130–7139
Hatton PJ, Cummins I, Cole DJ, Edwards R (1999) Glutathione transferases involved in herbicide detoxification in the leaves of Setaria faberi (giant foxtail). Plant Physiol 105:9–16
Hayes JD, Pulford DJ (1995) The glutathione S-transferase supergene family: regulation of GST and the contributions of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Bio 30:445–600
Hornby JAT, Luo J-K, Stevens JM, Wallace LA, Kaplan W, Armstrong RN, Dirr HW (2000) Equilibrium folding of dimeric class í glutathione transferases involves a stable monomeric. Interm Biochem 39:12336–12344
Jiao M, Li H-T, Chen J, Minton AP, Liang Y (2010) Attractive protein-polymer interactions markedly alter the effect of macromolecular crowding on protein association equilibria. Biophys J 99:914–923
Kanaoka Y, Ago H, Inagaki E, Nanayama T, Miyano M, Kikuno R, Fujii Y, Eguchi N, Toh H, Urade Y, Hayaishi O (1997) Cloning and crystal structure of hematopoietic prostaglandin D synthase. Cell 90:1085–1095
Liu P, Avramova LV, Park C (2009) Revisiting absorbance at 230 nm as a protein unfolding probe. Anal Biochem 389:165–170
Minton AP (2000) Implications of macromolecular crowding for protein assembly. Curr Opin Struct Biol 10:34–39
Minton AP (2005) Models for excluded volume interaction between an unfolded protein and rigid macromolecular cosolutes: macromolecular crowding and protein stability revisited. Biophys J 88:971–985
Monterroso B, Minton AP (2007) Effect of high concentration of inert cosolutes on the refolding of an enzyme: carbonic anhydrase B in sucrose and ficoll 70. J Bio Chem 282(46):33452–33458
Mora S, Elodi P (1968) Investigation of the near and far ultraviolet denaturation difference spectra of dehydrogenases. Eur J Biochem 5:574–582
Norris MG, Malys N (2011) What is the true enzyme kinetics in the biological system? An investigation of macromolecular crowding effect upon enzyme kinetics of glucose-6-phosphate dehydrogenase. Biochem Biophys Res Com 405:388–392
Ojopagogo YA, Adewale IO, Afolayan A (2013) Preliminary studies on the renaturation of denatured catfish C. gariepinus glutathione transferase. Fish Phys Biochem 39:1657–1663
Quasim A, Taha M (2013) Investigation of the mechanism of protein denaturation by guanidine hydrochloride-induced dissociation of inhibitor-protease complexes. Protein Pept Lett 20(2):187–191
Ralston GB (2005) Effects of “crowding” in protein solutions. J Chem Ed 67:857–860
Ranson H, Rossiter L, Ortelli F, Jensen B, Wang X, Roth CW, Collins FH, Hemingway J (2001) Identification of a novel class of insect glutathione S- transferases involved in DDT resistance in the malaria vector, Anopheles gambiae. Biochem J 359:295–304
Rivas G, Ferrone F, Herzfeld J (2003) Life in a crowded world. Eur Mol Bio Org 5:23–27
Royer CA, Mann CJ, Matthews CR (1993) Resolution of fluorescence equilibrium unfolding profile of trp aporepressor using simple tryptophan mutant. Protein Sci 2:1844–1853
Sacchetta PS, Pennelli A, Bucciarelli T, Cornelio L, Mmicarelli F, Miranda M, Ilio CD (1999) Multiple unfolded states of glutathione transferases bbGSTP1-1 by guanidinium chloride. Arch Biochem Biophys 369:100–106
Saxena M, Henderson GB (1995) ATP-dependent Efflux of 2,4-Dinitrophenyl-S-glutathione: Properties of two distinct transport systems in inside-out vesicles from l1210 cells and a variant subline with altered efflux of methotrexate and cholate. J Biol Chem 270(10):5312–5319
Sheehan D, Meade G, Foley VM, Dowd CA (2001) Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem J 360:1–16
Stevens JM, Hornby JAT, Armstrong RN, Dirr HW (1998) Class sigma glutathione transferase unfolds via a dimeric and a monomeric intermediate: impact of subunit interface on conformational stability in the superfamily. Biochemistry 37(44):15534–15541
Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412
Wei J, Dobnikar J, Curk T, Song F (2016) The effect of attractive interactions and macromolecular crowding on crystallins association. PLoS ONE 11(3):1–13
Wilcox AE, Micaela AL, Kristin MS (2016) Effects of macromolecular crowding on alcohol dehydrogenase activity are substrate-dependent. Biochemistry. https://doi.org/10.1021/acs.biochem.6b00257
Yu SJ (1996) Insect glutathione S-transferases. Zoo Stud 35:9–19
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OA thanks the Tertiary Education Trust Fund (TETfund) of Nigeria for providing postgraduate research funding for the laboratory work.
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Ogunmoyole, T., Fodeke, A.A. & Adewale, I.O. Denaturation studies of Clarias gariepinus glutathione transferase in dilute and crowded solutions. Eur Biophys J 48, 789–801 (2019). https://doi.org/10.1007/s00249-019-01405-z
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DOI: https://doi.org/10.1007/s00249-019-01405-z