Soybean proteins are limited by their low contents of methionine and cysteine. Herein, 7S globulin accumulation was reduced using RNA interference to silence CG-β-1 expression, and the content of the A2B1a subunit was largely increased under the soybean seed-specific oleosin8 promoter. The results showed that the sulfur-containing amino acid content in soybean seeds drastically improved, reaching 79.194 nmol/mg, and the 11S/7S ratio had a 1.89-fold increase compared to the wild-type acceptor. The secondary structures of 11S globulin were also altered, and the β-sheet content increased with decreasing β-turn content, which was confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy and circular dichroism analysis. Our findings suggested that raising the accumulation of 11S glycinin at the expense of reducing the content of 7S globulin is an attractive and precise engineering strategy to increase the amount of sulfur-containing amino acids, and soybean proteins with A2B1a subunits of 11S isolates improved, and β-subunits of 7S fractions reduced simultaneously might be an effective new material for food production.

The influences of multi-frequency countercurrent S-type ultrasound (MFSU), with various frequency modes, on lysinoalanine (LAL) formation and conformational characteristics of rice dreg protein isolates (RDPI) were investigated. The ultrasonic operating mode with dual-frequency combination (20/40 kHz) indicated lower LAL content and higher protein dissolution rate of RDPI compared with that of other ultrasound operating modes. Under the dual-frequency ultrasound mode of 20/40 kHz, acoustic power density of 60 W/L, time of 20 min, and temperature of 35 °C, the relative reduction rate of LAL of RDPI reached the highest with its value of 26.95%, and the protein dissolution rate was 71.87%. The changes in chemical interactions between protein molecules indicated that hydrophobic interactions and disulfide bonds played a considerable role in the formation of LAL of RDPI, especially the reduction of g-g-g and g-g-t disulfide bond. Alterations in microstructure showed that ultrasonication loosened the protein structure and created more uniform protein fragments of RDPI. In conclusion, using MFSU in treating RDPI was an efficacious avenue for minimizing LAL content and modifying the conformational characteristics of RDPI.

The influences of dual-frequency slit ultrasound (DFSU) pretreatment with various working parameters on the enzymolysis efficiency and conformational characteristics of corn gluten meal (CGM) were studied. Results indicated that under the conditions of ultrasonic power density of 80 W/L, time of 30 min, ultrasonic intermittent ratio of 5:2 s/s, temperature of 30 °C, and substrate concentration of 50 g/L, the relative enzymolysis efficiency (REE) of CGM reached a maximum of 21.05%, and the protein dissolution rate was 68.50%. In addition, ultrasonication had considerable impact on the conformation of CGM and consequently improved the susceptibility to alcalase proteolysis. Changes in free sulfhydryl (SHF) and disulfide bonds (SS) groups indicated spatial conformation of CGM was altered following sonication (sonochemical) treatment. Fourier Transform Infrared Spectrum (FITR) analysis showed a reduction in α-helix and β-turn content; and an increase in β-sheet and random coil content of CGM. Alterations in the particle size, particle size distribution, microstructure and surface roughness (Ra, Rq) indicated generation of smaller and more uniform protein fragments of CGM by sonochemical pretreatment. The proposed mechanism of sonicated CGM was elaborated. Our findings suggest that using DFSU in pretreating CGM may be an efficacious approach to enhance proteolysis.

Recent computational simulations on protein-ligand binding/unbinding have precisely been uncovering the ligand-binding process at the atomic level. In the process, the non-specific binding of ligands to the target site is suggested to occur before binding to the target. We in this study analyzed the conformations of ligands under the non-specific binding on a protein surface to figure out the differences in the conformational characteristics in aqueous solution using the 55-ns molecular dynamic simulation. As for the protein surface, we constructed an artificial β-sheet, composed of poly-alanine residues (Ala-sheet). For the ligands, the four α-thrombin inhibitors possessing two scaffolds with distinct hydrophobicity profiles were used. During the simulation, all the inhibitors kept interaction with Ala-sheet and had the limited conformational fluctuations compared with in aqueous solution. The representative conformations obtained from the cluster analysis showed that two of hydrophobic inhibitors adopted the extended conformations in aqueous solution and also on Ala-sheet. For the other two hydrophilic inhibitors, the conformations in aqueous solution adopted the bent conformation with two terminal hydrophobic rings closely packed. On Ala-sheet, contrarily, the two hydrophobic rings were open and took the extended conformations, which were placed on the sheet as a foothold. The charged moieties in the hydrophilic inhibitors were protruded into aqueous environment with the extended conformation. The conformational characteristics of the inhibitors in aqueous solution and Ala-sheet varied likely by chemical features or structures of the inhibitors, but each was considered to be physicochemically reasonable.