Crystal Structure of Bovine Alpha-Chymotrypsin in Space Group P65

Marshall, A.C.; Keiller, Benjamin G.; Pederick, Jordan L.; Abell, Andrew D.; Bruning, John B.

doi:10.3390/cryst8120460

Cited by 4 publications

(2 citation statements)

References 17 publications

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“…Therefore, it could be assumed that the interaction of ACT with EG might vary from other proteins. In addition, ACT consists of three polypeptide chains of different lengths connected by disulfide bonds (Figure S1D). , The solvation of interfacial regions of the chains may be different from the solvation properties of single-chain globular proteins. Within the limitations of the present MD simulation studies, we could not quantitatively analyze these solvation effects and their plausible effect on the hydration changes in the protein.…”

Section: Discussionmentioning

confidence: 99%

Cryo vs Thermo: Duality of Ethylene Glycol on the Stability of Proteins

2020

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Osmolytes are known to stabilize proteins under stress conditions. Thermal denaturation studies on globular proteins (β-lactoglobulin, cytochrome c, myoglobin, α-chymotrypsin) in the presence of ethylene glycol (EG), a polyol class of osmolyte, demonstrate a unique property of EG. EG stabilizes proteins against cold denaturation and destabilizes them during heat-induced denaturation. Further, chemical denaturation experiments performed at room temperature and at a sub-zero temperature (−10 °C) show that EG stabilizes the proteins at subzero temperature but destabilizes them at room temperature. The experiments carried out in the presence of glycerol, however, showed that glycerol stabilizes proteins against all of the denaturing conditions. This differential effect has not been reported for any other polyol class of osmolyte and might be specific to EG. Moreover, molecular dynamics simulations of all of the four proteins were carried out at three different temperatures, 240, 300, and 340 K, in the absence and presence of EG (20 and 40%). The results suggest that EG preferably accumulates around the hydrophobic residues and reduces the hydrophobic hydration of the proteins at a low temperature leading to stabilization of the proteins. At 340 K, the preferential hydration of the proteins is significantly reduced and the preferential binding of EG destabilizes the proteins like common denaturants.

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Section: Discussionmentioning

confidence: 99%

Cryo vs Thermo: Duality of Ethylene Glycol on the Stability of Proteins

2020

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“…The degree of protein purity according to the supplier (Sigma-Aldrich) is 92% for the batch used. Chymotrypsin comprises three polypeptide chains: A chain (13 amino acid residues, 1.25kDa), B chain (131 amino acid residues, 13.9 kDa) and C chain (97 amino acid residues, 10.1 kDa), linked by disulphide bridges 25 . Therefore, B and C chain are expected to be observed in SDS-PAGE under reducing conditions within the range of 6-14 kDa, as reflected in Figure 64.…”

Section: Levels Of Uncertainty Found and Mitigation Of Potential Issuesmentioning

confidence: 99%

Report on EFSA project OC/EFSA/GMO/2017/01 “In vitro protein digestibility” (Allergestion)

Mackie

Dupont

Torcello‐Gómez

et al. 2019

EFSA Supporting Publications

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The primary objective of the assessment of novel proteins is to evaluate whether they are safe to consume, including potential allergenicity. As part of a suite of assessments, the in vitro digestion of protein has been seen as a useful exercise. Thus, in line with the guidance offered by the EFSA GMO Panel we are using an early phase and a late phase gastric simulation as well as a simulation of the infant gastric compartment, all followed by intestinal phases. These digestion scenarios were used with a panel of 10 proteins from plant and animal origin that were proteins with distinct allergenic potential. The results from the SDS‐PAGE and densitometry show significant and mainly expected differences between the different digestion scenarios. The milk proteins were fully digested in the intestinal phase but the BLG was largely resistant to pepsin. In contrast, the egg proteins showed significant persistence except under late phase conditions. For the plant proteins, KTI and ConA were largely resistant to all conditions whereas LIP and AP were only resistant to infant conditions. Similarly, Ara h 1 showed some resistance to infant gastric conditions. The LC‐MS analysis of peptides was able to highlight a number of clusters where differences were seen between the digestion scenarios and these could in some cases be mapped onto the primary sequence and where relevant compared with known allergenic epitopes. Under the different digestion scenarios, we were able to show significant differences in the persistence of peptides larger than 9 amino acids and significant overlap of abundant peptides from early phase intestinal digestion and known epitopes for a number of proteins. Although, linking these differences to immunological responses (epitope mapping) still seems to be quite challenging, there are clear differences between scenarios and strong potential for improved risk assessment.

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