Insertion of antihypertensive peptides in acidic subunit from amaranth 11S induces contrasting effects in stability

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.

Foaming and Structural Studies on the Acidic Subunit of Amaranth 11S Globulin Modified with Antihypertensive Peptides as a Function of pH and Ionic Strength

“…The results on the elution properties of proteins are similar to those previously reported by [ 13 ] who reported that AAC and AACM.3.4 were eluted at low imidazole concentration and the rest of the proteins were eluted at higher imidazole concentration. This may suggest that the histidine tag of the AAC and AACM3.4 may be hidden Results suggested that the structure adopted by proteins AAC and AACM3.4 made the histidine tag unavailable or somewhat hidden, and that is why they elute at low concentrations of imidazole.…”

“…The maximum emission wavelength (λmax) of all proteins was observed from 337 to 350 nm at different pH conditions ( Table 1 ) which is characteristic of a polar environment. It has been reported that when tryptophan (Trp) residues are in a polar environment λmax is higher than 330 nm [ 20 ]; similar results were obtained by [ 13 ] who reported λmax between the range 334–341 nm at pH 7.5 for all proteins.…”

“…In our research group, the acidic polypeptide of amaranthine subunit (AAC) was modified by introducing four in tandem VY antihypertensive peptides (VYVYVYVY) at each variable region in order to know if the structural and conformation changes adopted by the modified proteins affected the possible antihypertensive effect [ 13 ].…”

“…The peptides were inserted at positions M1-E2 (AACM.1), R120-G121 (AACM.2), R199-E200 (AACM.3), and R272-L273 (AACM.4). Another construct was made by inserting VYVYVYVY at R199-E200 and RIPP sequence at the C-terminus position, which was identified as AACM.3.4 [ 13 , 14 ]. Our findings revealed that the insertion in the variable region I stabilized the protein thermally and chemically, but it affected the inhibitory activity of the angiotensin-converting enzyme in vitro.…”

“…The modified proteins AACM.2, AACM.3, and AACM.4 showed 8 times more antihypertensive activity than AAC and AACM.1, while AACM.3.4 showed 10 times more antihypertensive activity than AAC and AACM.1. So, the insertion of bioactive peptides in variable regions of the protein can induce conformational changes that may also alter its biological activity [ 13 ].…”