Abstract
β-Lactoglobulin is one of the most notable representatives of the lipocalin family of proteins that are mainly involved in the transportation of small hydrophobic molecules. Therefore, the conformation stability of these proteins represents an important step for several industrial applications. In this study, the β-lactoglobulin (β-lg) conformation stability in solution was studied as a function of the protein concentration, pH, NaCl and temperature when the protein concentration is significantly higher in the buffer solution. Using small-angle X-ray and neutron scattering methods (SANS and SAXS), it was shown that β-lg forms dimeric structures in 50 mM HEPES buffer under various conditions of the medium. At pH 5.9, β-lg preserves its dimeric form in the high protein concentration range of 10–40 mg/mL. However, the conformation swelled with the addition of 50 mM NaCl in the system with 20 mg/mL β-lg concentration. Further increase of NaCl concentration leads to a decrease of the radius of gyration of the β-lg dimer. At pH below 5.9 where the protein is positively charged, β-lg dimer preserves its conformation, and only an increase of the radius of gyration was attested by SAXS as the acidity of the medium increased to pH 3.6. This supports and adds to the findings presented above regarding the stability of β-lg dimer in 50 mM HEPES buffer in D2O. Furthermore, at neutral and alkaline pH, the values of both radius of gyration and maximum particle dimension decrease by increasing the pH of the medium.
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References
Allec N, Choi M, Yesupriya N, Szychowski B, White MR, Kann MG, Garcin ED, Daniel M-Ch, Badano A (2015) Small-angle X-ray scattering method to characterize molecular interactions: proof of concept. Sci Rep 5:12085. https://doi.org/10.1038/srep12085
Anandakrishnan R, Aguilar B, Onufriev AV (2012) H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulation. Nucleic Acids Res 40(W1):W537–W541
Anghel L, Radulescu A, Erhan RV (2018) Structural aspects of human lactoferrin in the iron-binding process studied by molecular dynamics and small-angle neutron scattering. Eur Phys J E 41:109. https://doi.org/10.1140/epje/i2018-11720-x
Báez GD, Moro A, Ballerini GA, Busti PA, Delorenzi NJ (2011) Comparison between structural changes of heat-treated and transglutaminase cross-linked beta-lactoglobulin and their effects on foaming properties. Food Hydrocolloid 25(7):1758–1765. https://doi.org/10.1016/j.foodhyd.2011.02.033
Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci USA 98:10037–10041. https://doi.org/10.1073/pnas.181342398
Barbiroli A, Beringhelli T, Bonomi F, Donghi D, Ferranti P, Galliano M, Iametti S, Maggioni D, Rasmussen P, Scanu S, Vilardo MC (2009) Bovine β-lactoglobulin acts as an acid-resistant drug carrier by exploiting its diverse binding regions. Biol Chem 391(1):21–32. https://doi.org/10.1515/bc.2010.008
Bauer R, Carrotta R, Rischel Ch, Øgendal L (2000) Characterization and isolation of intermediates in beta-lactoglobulin heat aggregation at high pH. Biophys J 79(2):1030–1038. https://doi.org/10.1016/S0006-3495(00)76357-0
Bello M, Pérez-Hernández G, Fernández-Velasco DA, Arreguín-Espinosa R, García E (2008) Energetics of protein homodimerization: effects of water sequestering on the formation of β-lactoglobulin dimer. Proteins 70:1475–1487
Cannon D, Eichhorn SJ, Donald AM (2016) Structure of spherulites in insulin, β-lactoglobulin, and amyloid β. ACS Omega 1(5):915–922
Collini M, Leo B, Baldini G, Monaco HL, Galliano M (2002) Probing protein aggregation by time-resolved fluorescence during b-lactoglobulin crystal growth. Eur Biophys J 31(2):111–117. https://doi.org/10.1007/s00249-002-0208-4
Crowther JM, Allison JR, Smolenski GA, Hodgkinson AJ, Jameson GB, Dobson RCJ (2018) The self-association and thermal denaturation of caprine and bovine β-lactoglobulin. Eur Biophys J 47(7):739–750. https://doi.org/10.1007/s00249-018-1300-8
Gottschalk M, Nilsson H, Roos H, Halle B (2003) Protein self-association in solution: the bovine β-lactoglobulin dimer and octamer. Protein Sci 12(11):2404–2411. https://doi.org/10.1110/ps.0305903
Grzyb J, Latowski D, Strzałka K (2006) Lipocalins—a family portrait. J Plant Physiol 163(9):895–915. https://doi.org/10.1016/j.jplph.2005.12.007
Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Gr 14(1):33–38. https://doi.org/10.1016/0263-7855(96)00018-5
Jeffries CM, Graewert MA, Blanchet CE, Langley DB, Whitten AE, Svergun DI (2016) Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments. Nat Protoc 11(11):2122–2153
Jung J-M, Savin G, Pouzot M, Schmitt Ch, Mezzenga R (2008) Structure of heat-induced β-lactoglobulin aggregates and their complexes with sodium-dodecyl sulfate. Biomacromol 9(9):2477–2486
Kazumasa S, Oobatake M, Goto Y (2001) Salt-dependent monomer–dimer equilibrium of bovine β-lactoglobulin at pH 3. Protein Sci 10(11):2325–2335. https://doi.org/10.1110/ps.17001
Kurpiewska K, Biela A, Loch JI, Świątek S, Jachimska B, Lewiński K (2018) Investigation of high pressure effect on the structure and adsorption of β-lactoglobulin. Colloid Surf B 161:387–393. https://doi.org/10.1016/j.colsurfb.2017.10.069
Le Bon CH, Taco N, Durand D (1999) Kinetics of aggregation and gelation of globular proteins after heat-induced denaturation. Macromolecules 32(19):6120–6127
Loupiac C, Bonetti M, Pin S, Calmettes P (2006) β-Lactoglobulin under high pressure studied by small-angle neutron scattering. BBA Proteins Proteom 1764(2):211–216. https://doi.org/10.1016/j.bbapap.2005.10.012
Lu Z, Pan F, Wang D, Campana M, Xu H, Tucker IM, Petkov JT, Webstere J, Lu JR (2016) Unusual surface and solution behaviour of keratin polypeptides. RSC Adv 6:105192–105201
Majhi PR, Ganta RR, Vanam RP, Seyrek E, Giger K, Dubin PL (2006) Electrostatically driven protein aggregation: β-lactoglobulin at low ionic strength. Langmuir 22(22):9150–9159
Mensi A, Choiset Y, Haertlé T, Reboul E, Borel P, Guyon C, de Lamballerie M, Chobert JM (2013) Interlocking of β-carotene in beta-lactoglobulin aggregates produced under high pressure. Food Chem 139(1–4):253–260. https://doi.org/10.1016/j.foodchem.2013.02.004
Moitzi Ch, Donato L, Schmitt Ch, Bovetto L, Gillies G, Stradner A (2011) Structure of β-lactoglobulin microgels formed during heating as revealed by small-angle X-ray scattering and light scattering. Food Hydrocolloid 25(7):1766–1774. https://doi.org/10.1016/j.foodhyd.2011.03.020
Moro A, Báez GD, Ballerini GA, Busti PA, Delorenzi NJ (2013) Emulsifying and foaming properties of β-lactoglobulin modified by heat treatment. Food Res Int 51(1):1–7. https://doi.org/10.1016/j.foodres.2012.11.011
Nicolai T, Britten M, Schmitt Ch (2011) β-Lactoglobulin and WPI aggregates: formation, structure and applications. Food Hydrocolloid 25(8):1945–1962. https://doi.org/10.1016/j.foodhyd.2011.02.006
Pérez OE, David-Birman T, Kesselman E, Levi-Tal S, Lesmes U (2014) Milk protein-vitamin interactions: formation of beta-lactoglobulin/folic acid nano-complexes and their impact on in vitro gastro-duodenal proteolysis. Food Hydrocolloid 38:40–47. https://doi.org/10.1016/j.foodhyd.2013.11.010
Petoukhov MV, Franke D, Shkumatov AV, Tria G, Kikhney AG, Gajda M, Gorba C, Mertens HDT, Konarev PV, Svergun DI (2012) New developments in the ATSAS program package for small-angle scattering data analysis. J Appl Crystallogr 45:342–350. https://doi.org/10.1107/S0021889812007662
Rubinson KA, Chen Y, Cress BF, Zhang F, Linhardt RJ (2016) Heparin’s solution structure determined by small-angle neutron scattering. Biopolymers 105(12):905–913
Rubinson KA (2017) Practical corrections for p(H, D) measurements in mixed H2O/D2O biological buffers. Anal Methods 9:2744–2750
Sauter A, Zhang F, Szekely NK, Pipich V, Sztucki M, Schreiber F (2016) Structural evolution of metastable protein aggregates in the presence of trivalent salt studied by (V)SANS and SAXS. J Phys Chem B 120(24):5564–5571
Schlehuber S, Skerra A (2005) Lipocalins in drug discovery: from natural ligand-binding proteins to ‘anticalins’. Drug Discov Today 10(1):23–33. https://doi.org/10.1016/S1359-6446(04)03294-5
Soloviev AG, Solovjeva TM, Ivankov OI, Soloviov DV, Rogachev AV, Kuklin AI (2017) SAS program for two-detector system: seamless curve from both detectors. J Phys Conf Ser 848:012020
Svergun DI (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 25:495–503
Svergun DI, Petoukhov MV, Koch MHJ (2001) Determination of domain structure of proteins from X-ray solution scattering. Biophys J 80:2946–2953
Taulier N, Chalikian TV (2001) Characterization of pH-induced transitions of β-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies. J Mol Biol 314(4):873–889. https://doi.org/10.1006/jmbi.2001.5188
Tavares GM, Croguennec T, Carvalho AF, Bouhallab S (2014) Milk proteins as encapsulation devices and delivery vehicles: applications and trends. Trends Food Sci Tech 37(1):5–20. https://doi.org/10.1016/j.tifs.2014.02.008
van den Akker CC, Schleeger M, Bonn M, Koenderink GH (2014) Structural basis for the polymorphism of β-lactoglobulin amyloid-like fibrils. In: Uversky VN, Lyubchenko YL (eds) Bio-nanoimaging. Academic Press, Boston, pp 333–343. https://doi.org/10.1016/B978-0-12-394431-3.00031-6
Zabelskii DV, Vlasov AV, Ryzhykau YuL, Murugova TN, Brennich M, Soloviov DV, Ivankov OI, Borshchevskiy VI, Mishin AV, Rogachev AV, Round A, Dencher NA, Büldt G, Gordeliy VI, Kuklin AI (2018) Ambiguities and completeness of SAS data analysis: investigations of apoferritin by SAXS/SANS EID and SECSAXS methods. J Phys Conf Ser 994:012017
Zhang L, Lu D, Liu Z (2008) How native proteins aggregate in solution: a dynamic Monte Carlo simulation. Biophys Chem 133(1–3):71–80
Zhang F, Roosen-Runge F, Skoda MWA, Jacobs MJ, Wolf M, Callow Ph, Frielinghaus H, Pipich V, Prévostf S, Schreibera F (2012) Hydration and interactions in protein solutions containing concentrated electrolytes studied by small-angle scattering. Phys Chem Chem Phys 14(7):2483–2493
Zhang J, Liu X, Subirade M, Zhou P, Liang L (2014) A study of multi-ligand beta-lactoglobulin complex formation. Food Chem 165:256–261. https://doi.org/10.1016/j.foodchem.2014.05.109
Acknowledgements
This work was supported by a grant framework of the Romanian Plenipotentiary at JINR-Dubna, within the JINR Theme no. 04-4-1121-2015/2020. The authors gratefully acknowledge the beamtime received at IBR-2 pulsed reactor, FLNP, JINR, Dubna, Russian Federation, beamline YuMO small angle neutron scattering spectrometer for the proposals nos. 2015-10-15-23-59-22 and 2016-10-15-20-20-30 and to O. Ivankov, V. Skoj and M. Rulev for providing assistance during the measurements. The experiments were performed on beamline BM29 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The authors are grateful to Structural biology Group and Local Contact at the ESRF for providing assistance in using the beamline.
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Anghel, L., Rogachev, A., Kuklin, A. et al. β-Lactoglobulin associative interactions: a small-angle scattering study. Eur Biophys J 48, 285–295 (2019). https://doi.org/10.1007/s00249-019-01360-9
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DOI: https://doi.org/10.1007/s00249-019-01360-9