Abstract
The insertion of peptides is a biotechnology tool widely used to improve the nutraceutical properties of proteins. Because the effect of these insertions in protein stability and function is difficult to predict, it should be determined experimentally. In this study, we created two variants of amarantin acidic subunit and analyzed them along with other four proteins reported previously. We measured their response against two destabilizing agents: temperature and urea. The six proteins presented the insertion of antihypertensive peptides (VYVYVYVY or RIPP) in the variable regions of the protein. We observed that their effect strongly depended on the site of the insertion. The insertion in the variable region I stabilized the protein both thermally and chemically, but it affected the inhibitory activity of the angiotensin-converting enzyme in vitro. In contrast, insertions in other three regions were severely destabilizing, producing molten globules. Our findings reveal that the insertion of bioactive peptides in variable regions of a protein can increase or decrease the protein’s thermal and chemical stability and that these conformational changes may also alter its final activity.
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Barba De La Rosa AP, Herrera-Estrella A, Utsumi S, Paredes-López O (1996) Molecular characterization, cloning and structural analysis of a cDNA encoding an amaranth globulin. J Plant Physiol 149:527–532. https://doi.org/10.1016/S0176-1617(96)80329-4
Bennion BJ, Daggett V (2003) The molecular basis for the chemical denaturation of proteins by urea. Proc Natl Acad Sci U S A 100:5142–5147. https://doi.org/10.1073/pnas.0930122100
Carrazco-Peña L, Osuna-Castro JA, De León-Rodríguez A, Maruyama N, Toro-Vazquez JF, Morales-Rueda JA, Barba de la Rosa AP (2013) Modification of solubility and heat-induced gelation of amaranth 11S globulin by protein engineering. J Agric Food Chem 6:3509–3516. https://doi.org/10.1021/jf3050999
Castro-Martínez C, Luna-Suárez S, Paredes-López O (2012) Overexpression of a modified protein from amaranth seed in Escherichia coli and effect of environmental conditions on the protein expression. J Biotechnol 158:59–67. https://doi.org/10.1016/j.jbiotec.2011.12.012
Chen S, Paredes-López O (1997) Isolation and characterization of the 11S globulin from amaranth seeds. J Food Biochem 21:53–65. https://doi.org/10.1111/j.1745-4514.1997.tb00224.x
Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D 66:12–21. https://doi.org/10.1107/S0907444909042073
Dehghan-Nayeri N, Rezaei-Tavirani M (2015) The interpretation of protein structure through relationship of melting point (Tm) and enthalpy of unfolding (ΔHU). IJAPBS 41:47–50
Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of coot. Acta Crystallogr D 66:486–501. https://doi.org/10.1107/S0907444910007493
Fiser A, Sali A (2003) ModLoop: automated modeling of loops in protein structures. Bioinformatics 19:2500–2501. https://doi.org/10.1093/bioinformatics/btg362
FitzGerald RJ, Murray BA, Walsh DJ (2004) Hypotensive peptides from milk proteins. J Nutr 134:980–988
Fu H, Grimsley G, Scholtz JM, Pace CN (2010) Increasing protein stability: importance of ΔCp and the denatured state. Protein Sci 19:1044–1052. https://doi.org/10.1002/pro.381
Glasser SP (2001) Hypertension syndrome and cardiovascular events. Postgrad Med 110:29–36. https://doi.org/10.1080/00325481.2001.11445493
Heinz DW, Baase WA, Dahlquist FW, Matthews BW (1993) How amino-acid insertions are allowed in an α-helix of T4 lysozyme. Nature 361:561–564. https://doi.org/10.1038/361561a0
Heinz DW, Baase WA, Zhang X-J, Blaber M, Dahlquist FW, Matthews BW (1994) Accommodation of amino acid insertions in an α-helix of T4 lysozyme: structural an thermodynamic analysis. J Mol Biol 236:869–886. https://doi.org/10.1006/jmbi.1994.1195
Källberg M, Wang H, Wang S, Peng J, Wang Z, Lu H, Xu J (2012) Template-based protein structure modeling using the RaptorX web server. Nat Protoc 7:1511–1522. https://doi.org/10.1038/nprot.2012.085
Kawasaki T, Seki E, Osajima K, Yoshida M, Asada K, Matsui T, Osajima Y (2000) Antihypertensive effect of valyl–tyrosine, a short chain peptide derived from sardine muscle hydrolyzate, on mild hypertensive subjects. J Hum Hypertens 14:519–523. https://doi.org/10.1038/sj.jhh.1001065
Keerati-u-rai M, Miriani M, Lametti S, Bonomi F, Corredig M (2012) Structural changes of soy proteins at the oil–water interface studied by fluorescence spectroscopy. Colloids Surf B: Biointerfaces 93:41–48. https://doi.org/10.1016/j.colsurfb.2011.12.002
Kim CS, Kamiya S, Sato T, Utsumi S, Kito M (1990) Improvement of nutritional value and functional properties of soybean glycinin by protein engineering. Protein Eng 3(8):725–731. https://doi.org/10.1093/protein/3.8.725
Krieger E, Joo K, Lee J, Lee J, Raman S, Thompson J, Tyka M, Baker D, Karplus K (2009) Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: four approaches that performed well in CASP8. Proteins 9:114–122. https://doi.org/10.1002/prot.22570
Lavinder JJ, Hari SB, Sullivan BJ, Magliery TJ (2009) High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering. J Am Chem Soc 131:3794–3795. https://doi.org/10.1021/ja8049063
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
Luna-Suárez S, Medina-Godoy S, Cruz-Hernández A, Paredes-López O (2008) Expression and characterization of the acidic subunit from 11S amaranth seed protein. Biotechnol J 3:209–219. https://doi.org/10.1002/biot.200700146
Luna-Suárez S, Medina-Godoy S, Cruz-Hernández A, Paredes-López O (2010) Modification of the amaranth 11S globulin storage protein to produce an inhibitory peptide of the angiotensin I converting enzyme, and its expression in Escherichia coli. J Biotechnol 148:40–247. https://doi.org/10.1016/j.jbiotec.2010.06.009
Martin SR, Schilstra MJ (2008) Circular dichroism and its application to the study of biomolecules. Methods Cell Biol 84:263–293. https://doi.org/10.1016/S0091-679X(07)84010-6
Maruyama Y, Maruyama N, Mikami B, Utsumi S (2004) Structure of the core region of the soybean beta-conglycinin alpha subunit. Acta Crystallogr D 60:289–297. https://doi.org/10.1107/S0907444903027367
Matoba N, Usui H, Fujita H, Yoshikawa M (1999) A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR mesenteric artery. FEBS Lett 452:181–184. https://doi.org/10.1016/S0014-5793(99)00587-6
Matsui T, Matsufuji H, Seki E, Osajima K, Nakashima M, Osajima Y (1993) Inhibition of angiotensin I-converting enzyme by Bacillus licheniformis alkaline protease hydrolyzates derived from sardine muscle. Biosci Biotechnol Biochem 57:922–925. https://doi.org/10.1271/bbb.57.922
Matsui T, Zhu XL, Watanabe K, Tanaka K, Kusano Y, Matsumoto K (2006) Combined administration of captopril with an antihypertensive Val–Tyr di-peptide to spontaneously hypertensive rats attenuates the blood pressure lowering effect. Life Sci 79:2492–2498. https://doi.org/10.1016/j.lfs.2006.08.013
Moon CP, Fleming KG (2011) Using tryptophan fluorescence to measure the stability of membrane proteins folded in liposomes. Methods Enzymol 492:189–211. https://doi.org/10.1016/B978-0-12-381268-1.00018-5
Morales-Camacho JI, Paredes-López O, Espinosa-Hernández E, Fernández-Velasco DA, Luna-Suárez S (2016) Expression, purification and thermal stability evaluation of an engineered amaranth protein. Electron J Biotechnol 22:44–51. https://doi.org/10.1016/j.ejbt.2016.04.001
Nakamura Y, Yamamoto N, Sakai K, Takano T (1995) Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme. J Dairy Sci 78:1253–1257. https://doi.org/10.3168/jds.S0022-0302(95)76745-5
Onishi K, Nobuyuki M, Yamada Y, Doyama N, Maruyama N, Utsumi S, Yoshikawa M (2004) Optimal designing of β-conglycinin to genetically incorporate RPLKPW, a potent anti-hypertensive peptide. Peptides 25:37–43. https://doi.org/10.1016/j.peptides.2003.11.006
Pace CN (1986) Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol 131:266–280. https://doi.org/10.1016/0076-6879(86)31045-0
Pace CN, Shirley BA (1990) In: Creighton TE (ed) Measuring the conformational stability of a protein, protein structure: a practical approach. IRL Press, Oxford, pp 311–330
Phillips K, de la Peña AH (2011) The combined use of thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization. Curr Protoc Mol Biol, Unit10.28. https://doi.org/10.1002/0471142727.mb1028s94
Renata K, Virginija D, Jurate M (2015) Association of neck circumference and high blood pressure in children and adolescents: a case–control study. BMC Pediatr 15:127. https://doi.org/10.1186/s12887-015-0444-2
Rosen LE, Connel KB, Marqsee S (2014) Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule. Proc Natl Acad Sci U S A 111:14746–14751. https://doi.org/10.1073/pnas.1410630111
Scalley-Kim M, Minard P, Baker D (2003) Low free energy cost of very long loop insertions in proteins. Protein Sci 12:197–206. https://doi.org/10.1110/ps.0232003
So PTC, Dong CY (2001) In: eLS. Wiley. Fluorescence spectrophotometry. Ltd, Chichester
Sonavane S, Chakrabarti P (2008) Cavities and atomic packing in protein structures and interfaces. PLoS Comput Biol 4:10.1371. https://doi.org/10.1371/journal.pcbi.1000188
Sreerama N, Woody RW (2003) Structural composition of βI- and βII-proteins. Protein Sci 12:384–388. https://doi.org/10.1110/ps.0235003
Tandang MR, Atsuta N, Maruyama N, Adachi M, Utsumi S (2005) Evaluation of the solubility and emulsifying property of soybean proglycinin and rapeseed procruciferin in relation to structure modified by protein engineering. J Agric Food Chem 53:8736–8744. https://doi.org/10.1021/jf050871y
Tandang-Silvas MR, Tecson-Mendoza EM, Mikami B, Utsumi S, Maruyama N (2011) Molecular design of seed storage proteins for enhanced food physicochemical properties. Annu Rev Food Sci Technol 2:59–73. https://doi.org/10.1146/annurev-food-022510-133718
Tandang-Silvas MR, Cabanos CC, Carrazco-Peña L, Barba de la Rosa AP, Osuna-Castro JA, Utsumi S, Mikami B, Maruyama N (2012) Crystal structure of a major seed storage protein, 11S proglobulin, from Amaranthus hypochondriacus: insight into its physico-chemical properties. Food Chem 135:819–826. https://doi.org/10.1016/j.foodchem.2012.04.135
Utsumi S, Maruyam N, Satoh R, Adachi M (2002) Structure-function relationships of soybean proteins revealed by using recombinant systems. Enzym Microb Technol 30:284–288. https://doi.org/10.1016/S0141-0229(01)00507-5
Vermeirssen V, Van Camp J, Decroos K, Van Wijmelbeke L, Verstraete W (2003) The impact of fermentation and in vitro digestion on the formation of angiotensin-I-converting enzyme inhibitory activity from pea and whey protein. J Dairy Sci 86:429–438. https://doi.org/10.3168/jds.S0022-0302(03)73621-2
Vetter IR, Baase WA, Heinz DW, Xiong J-P, Snow S, Matthews BW (1996) Proteins structural plasticity exemplified by insertion and deletion mutants in T4 lysozyme. Protein Sci 5:2399–2415. https://doi.org/10.1002/pro.5560051203
Whitmore L, Wallace BA (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32:668–673. https://doi.org/10.1093/nar/gkh371
World Health Organization (2009) Mortality and burden of disease attributable to selected major risks. WHO Press, World Health Organization, Geneva
Wu J, Yang JT, Wu CS (1992) Beta-II conformation of all-beta proteins can be distinguished from unordered form by circular dichroism. Anal Biochem 200:359–364. https://doi.org/10.1016/0003-2697(92)90479-Q
Zhang R, Monsma F (2010) Fluorescence-based thermal shift assays. Curr Opin Drug Discover Devel 13:389–402
Acknowledgements
We thank Dr. V.E. López and M. L. Llamas-García for their technical assistance.
Funding
This work was supported by the Consejo Nacional de Ciencia y Tecnología [Grant no. 103399, 256,478, 270,245] (fellowship PhD for JIMC and EEH) as well as by the Secretaría de Investigación y Posgrado-IPN [Grant nos. 20144006, 20171468]
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Morales-Camacho, J.I., Espinosa-Hernández, E., Fernández-Velasco, D.A. et al. Insertion of antihypertensive peptides in acidic subunit from amaranth 11S induces contrasting effects in stability. Appl Microbiol Biotechnol 102, 9595–9606 (2018). https://doi.org/10.1007/s00253-018-9300-2
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DOI: https://doi.org/10.1007/s00253-018-9300-2