Biophysical Characterization of Temperature-Dependent Structural Modifications in β-Lactoglobulin during Tryptic Hydrolysis
DOI:
https://doi.org/10.5281/zenodo.11266062Keywords:
Proteolysis, Protein secondary structure, FTIR spectroscopyAbstract
Determination of peptide fragments of proteins upon proteolysis process is crucial in many fields, including biotechnology, proteomics and food industry. Herein, our aim was to compare the structural changes of intact protein during enzymatic hydrolysis at various temperatures by using biophysical approaches. Proteolysis of β-Lactoglobulin (β-LG) was carried out with trypsin, and the changes were followed in situ during proteolysis at 37°C and 45°C by using Fourier transform infrared (FTIR) spectroscopy. Dynamical response of β-LG to enzymatic attack was identified on the molecular level. The IR signals of protein secondary structures decreased while carboxylate group signals arising from liberated products of proteolytic reaction increased in the 1605-1580 cm-1 range to the different extent. FTIR data revealed that the degree of protein changes and liberated products varies greatly based on the reaction temperature. This study demonstrates the potentiality of FTIR spectroscopy for in situ tracking of protein hydrolysis at various conditions.
References
Barth, A. (2000). The infrared absorption of amino acid side chains. Progress in Biophysics and Molecular Biology, 74(3–5), 141–173. https://doi.org/10.1016/S0079-6107(00)00021-3
Broersen, K. (2020). Milk processing affects structure, bioavailability and immunogenicity of β-lactoglobulin. Foods, 9(7), 874. https://doi.org/10.3390/foods9070874
Cheison, S. C., A, M.-Y. L., Leeb, E., & Kulozik, U. (2011). Hydrolysis of β-lactoglobulin by trypsin under acidic pH and analysis of the hydrolysates with MALDI–TOF–MS/MS. Food Chemistry, 125(4), 1241–1248. https://doi.org/10.1016/j.foodchem.2010.10.042
Derenne, A., Vandersleyen, O., & Goormaghtigh, E. (2014). Lipid quantification method using FTIR spectroscopy applied on cancer cell extracts. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1841(8), 1200–1209. https://doi.org/10.1016/J.BBALIP.2013.10.010
Errico, S., Moggio, M., Diano, N., Portaccio, M., & Lepore, M. (2023). Different experimental approaches for Fourier-transform infrared spectroscopy applications in biology and biotechnology: A selected choice of representative results. Biotechnology and Applied Biochemistry, 70(3), 937–961. https://doi.org/10.1002/bab.2411
Fabian, H., & Mäntele, W. (2006). Infrared spectroscopy of proteins. In J. M. Chalmers & P. R. Griffiths (Eds.), Handbook of Vibrational Spectroscopy (pp. 3399–3425). John Wiley & Sons, Ltd. https://doi.org/doi.org/10.1002/0470027320.s8201
Galvão, C. M., Silva, A. F., Custódio, M. F., Monti, R., & Giordano, R. L. (2001). Controlled hydrolysis of cheese whey proteins using trypsin and α-chymotrypsin. Applied Biochemistry and Biotechnology, 91, 761-776. https://doi.org/10.1385/ABAB:91-93:1-9:761
Golovanov, A., Güler, G., & Vorob’ev, M. M. (2024). Modification of protein micelles by limited hydrolysis of peptide bonds: A model of the sequential degradation of β-casein micelles. INEOS OPEN - Journal of Nesmeyanov Institute of Organoelement Compounds of the Russian Academy of Sciences, 6(2), 44–48. https://doi.org/10.32931/io2313a
Güler, G., Acikgoz, E., Karabay Yavasoglu, N. Ü., Bakan, B., Goormaghtigh, E., & Aktug, H. (2018). Deciphering the biochemical similarities and differences among mouse embryonic stem cells, somatic and cancer cells using ATR-FTIR spectroscopy. The Analyst, 143(7), 1624–1634. https://doi.org/10.1039/c8an00017d
Güler, G., Acikgoz, E., Mukhtarova, G., & Oktem, G. (2024). Biomolecular fingerprints of the effect of zoledronic acid on prostate cancer stem cells: Comparison of 2D and 3D cell culture models. Archives of Biochemistry and Biophysics, 753, 109920. https://doi.org/10.1016/j.abb.2024.109920
Güler, G., Džafić, E., Vorob’Ev, M. M., Vogel, V., & Mäntele, W. (2011). Real time observation of proteolysis with Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy: Watching a protease eat a protein. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy, 79(1), 104–111. https://doi.org/10.1016/j.saa.2011.01.055
Güler, G., Gärtner, R. M., Ziegler, C., & Mäntele, W. (2016). Lipid-protein interactions in the regulated betaine symporter BetP probed by infrared spectroscopy. The Journal of Biological Chemistry, 291(9), 4295–4307. https://doi.org/10.1074/jbc.M114.621979
Guler, G., Guven, U., & Oktem, G. (2019). Characterization of CD133(+)/CD44(+) human prostate cancer stem cells with ATR-FTIR spectroscopy. The Analyst, 144(6), 2138–2149. https://doi.org/10.1039/c9an00093c
Güler, G., Vorob’ev, M. M., Vogel, V., & Mäntele, W. (2016). Proteolytically-induced changes of secondary structural protein conformation of bovine serum albumin monitored by Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy. Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy, 161, 8–18. https://doi.org/10.1016/j.saa.2016.02.013
Iametti, S., Rasmussen, P., Frøkiaer, H., Ferranti, P., Addeo, F., & Bonomi, F. (2002). Proteolysis of bovine b-lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments with low immunoreactivity. European Journal of Biochemistry, 269(5), 1362–1372. https://doi.org/10.1046/j.1432-1033.2002.02769.x
Júnior, E. C. S., Santos, M. P. F., Sampaio, V. S., Ferrão, S. P. B., Fontan, R. C. I., Bonomo, R. C. F., & Veloso, C. M. (2020). Hydrolysis of casein from different sources by immobilized trypsin on biochar: Effect of immobilization method. Journal of Chromatography B, 1146, 122124. https://doi.org/10.1016/j.jchromb.2020.122124
Korkmaz, F., Ressl, S., Ziegler, C., & Mäntele, W. (2013). K(+)-induced conformational changes in the trimeric betaine transporter BetP monitored by ATR-FTIR spectroscopy. Biochimica et Biophysica Acta, 1828, 1181–1191. https://doi.org/10.1016/j.bbamem.2013.01.004
Korkmaz, F., van Pee, K., & Yildiz, Ö. (2015). IR-spectroscopic characterization of an elongated OmpG mutant. Archives of Biochemistry and Biophysics, 576, 73–79. https://doi.org/10.1016/j.abb.2015.04.010
Kristoffersen, K. A., Liland, K. H., Böcker, U., Wubshet, S. G., Lindberg, D., Horn, S. J., & Afseth, N. K. (2019). FTIR-based hierarchical modeling for prediction of average molecular weights of protein hydrolysates. Talanta, 205, 120084. https://doi.org/10.1016/J.TALANTA.2019.06.084
Markoska, T., Daniloski, D., Vasiljevic, T., & Huppertz, T. (2021). Structural changes of β-casein induced by temperature and pH analysed by nuclear magnetic resonance, fourier-transform infrared spectroscopy, and chemometrics. Molecules, 26, 7650. https://doi.org/10.3390/molecules26247650
Oliveira, K. M., Valente-Mesquita, V. L., Botelho, M. M., Sawyer, L., Ferreira, S. T., & Polikarpov, I. (2001). Crystal structures of bovine beta-lactoglobulin in the orthorhombic space group C222(1). Structural differences between genetic variants A and B and features of the Tanford transition. European Journal of Biochemistry, 268(2), 477–483. https://doi.org/10.1046/j.1432-1033.2001.01918.x
Olsen, J. V, Ong, S.-E., & Mann, M. (2004). Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Molecular & Cellular Proteomics, 3(6), 608–614. https://doi.org/10.1074/mcp.T400003-MCP200
Origone, A. L., Hissi, E. G. V., Liggieri, C. S., Camí, G. E., Illanes, A., & Barberis, S. E. (2024). Effect of organic solvents on the activity, stability and secondary structure of asclepain cI, using FTIR and molecular dynamics simulations. The Protein Journal, https://doi.org/10.1007/s10930-024-10182-4
Pirutin, S. K., Jia, S., Yusipovich, A. I., Shank, M. A., Parshina, E. Y., & Rubin, A. B. (2023). Vibrational spectroscopy as a tool for bioanalytical and biomonitoring studies. International Journal of Molecular Sciences, 24(8), 6947. https://doi.org/10.3390/ijms24086947
Poulsen, N. A., Eskildsen, C. E., Akkerman, M., Johansen, L. B., Hansen, M. S., Hansen, P. W., Skov, T., & Larsen, L. B. (2016). Predicting hydrolysis of whey protein by mid-infrared spectroscopy. International Dairy Journal, 61, 44–50. https://doi.org/10.1016/J.IDAIRYJ.2016.04.002
Sen, I., Bozkurt, O., Aras, E., Heise, S., Brockmann, G. A., & Severcan, F. (2015). Lipid profiles of adipose and muscle tissues in mouse models of juvenile onset of obesity without high fat diet induction: a Fourier transform infrared (FT-IR) spectroscopic study. Applied Spectroscopy, 69(6), 679-688. https://doi.org/10.1366/14-07443
Toldrá, F., & Mora, L. (2021). Enzymatic mechanisms for the generation of bioactive peptides. In Fidel Toldrá & Leticia Mora (Eds.), Biologically Active Peptides, From Basic Science to Applications for Human Health (pp. 27–46). Academic Press. https://doi.org/10.1016/C2019-0-02469-7
Vorob’ev, M. M. (2022). Modeling of proteolysis of β-lactoglobulin and β-casein by trypsin with consideration of secondary masking of intermediate polypeptides. International Journal of Molecular Sciences, 23(15), 8089. https://doi.org/10.3390/ijms23158089
Vorob’ev, M. M., Açıkgöz, B. D., Güler, G., Golovanov, A. V., & Sinitsyna, O. V. (2023). Proteolysis of micellar β-casein by trypsin: secondary structure characterization and kinetic modeling at different enzyme concentrations. International Journal of Molecular Sciences, 24(4), 3874. https://doi.org/10.3390/IJMS24043874
Vorob’ev, M. M., Strauss, K., Vogel, V., & Mäntele, W. (2015). Demasking of peptide bonds during tryptic hydrolysis of β-casein in the presence of ethanol. Food Biophysics, 10(3), 309–315. https://doi.org/10.1007/s11483-015-9391-6
Vorob’ev, M. M., Vogel, V., & Mäntele, W. (2013). Demasking rate constants for tryptic hydrolysis of β-casein. International Dairy Journal, 30(1), 33-38. https://doi.org/10.1016/j.idairyj.2012.12.002
Vorob’ev, M. M., Vogel, V., Güler, G., & Mäntele, W. (2011). Monitoring of demasking of peptide bonds during proteolysis by analysis of the apparent spectral shift of intrinsic protein fluorescence. Food Biophysics, 6(4), 519–526. https://doi.org/10.1007/s11483-011-9234-z
Wei, J., Wagner, S., Maclean, P., Brophy, B., Cole, S., Smolenski, G., Carlson, D. F., Fahrenkrug, S. C., Wells, D. N., & Laible, G. (2018). Cattle with a precise, zygote-mediated deletion safely eliminate the major milk allergen beta-lactoglobulin. Scientific Reports, 8, 7661. https://doi.org/10.1038/s41598-018-25654-8
Yang, H., Yang, S., Kong, J., Dong, A., & Yu, S. (2015). Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy. Nature Protocols, 10(3), 382–396. https://doi.org/10.1038/nprot.2015.024
Yang, S., Zhang, Q., Yang, H., Shi, H., Dong, A., Wang, L., & Yu, S. (2022). Progress in infrared spectroscopy as an efficient tool for predicting protein secondary structure. International Journal of Biological Macromolecules, 206, 175–187. https://doi.org/10.1016/j.ijbiomac.2022.02.104
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