ea33b83db9ad108378991cc832c6a1f6 ijms-26-01150-v2.pdf 1004759c75d9321d04e8434cc2dd7e14f468b16d ijms-26-01150-v2.pdf f26c929b5f4b2cf2acecfc72533aef0de0d43ae2fbebce08638b42e8377cc698 ijms-26-01150-v2.pdf Title: Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure–Function Characterization of the Amaranthus caudatus Species Subject: Protease inhibitors are biomolecules with growing biotechnological and biomedical relevance, including those derived from plants. This study investigated strong trypsin inhibitors in quinoa, amaranth, and lupine seeds, plant grains traditionally used in Andean South America. Amaranth seeds displayed the highest trypsin inhibitory activity, despite having the lowest content of aqueous soluble and thermostable protein material. This activity, directly identified by enzymatic assay, HPLC, intensity-fading mass spectrometry (IF-MS), and MS/MS, was attributed to a single protein of 7889.1 Da, identified as identical in Amaranthus caudatus and A. hybridus, with a Ki of 1.2 nM for the canonical bovine trypsin. This form of the inhibitor, which is highly homogeneous and scalable, was selected, purified, and structurally–functionally characterized due to the high nutritional quality of amaranth seeds as well as its promising agriculture–biotech–biomed applicability. The protein was crystallized in complex with bovine trypsin, and its 3D crystal structure resolved at 2.85 Å, revealing a substrate-like transition state interaction. This verified its classification within the potato I inhibitor family. It also evidenced that the single disulfide bond of the inhibitor constrains its binding loop, which is a key feature. Cell culture assays showed that the inhibitor did not affect the growth of distinct plant microbial pathogen models, including diverse bacteria, fungi, and parasite models, such as Mycoplasma genitalium and Plasmodium falciparum. These findings disfavour the notion that the inhibitor plays an antimicrobial role, favouring its potential as an agricultural insect deterrent and prompting a redirection of its functional research. Keywords: plant trypsin inhibitors; quinoa; amaranth; lupine seeds; Amaranthus hybridus; Amaranthus caudatus; HPLC; MS and X-ray analysis; structure–function characterisation; plant defence Author: Martha Hernández de la Torre, Giovanni Covaleda-Cortés, Laura Montesinos, Daniela Covaleda, Juan C. Ortiz, Jaume Piñol, José M. Bautista, J. 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Natural ATSI Destination: subsection.2.2 Item: Title: Determination of Inhibitory Activities, Ki Value, Number of Cysteines, and Top-Down MS Sequencing Analysis Destination: subsection.2.3 Item: Title: Formation and Isolation of the ATSI-Bovine Trypsin Complex Destination: subsection.2.4 Item: Title: Three-Dimensional Crystal Structure Analysis of the ATSI-bTrypsin Complex Destination: subsection.2.5 Item: Title: Structure Function Details of the ATSI-Trypsin Complex Destination: subsection.2.6 Item: Title: Assaying the Capabilities of ATSI to Act as an Antimicrobial in Cell Cultures Destination: subsection.2.7 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Materials Destination: subsection.4.1 Item: Title: Methods Destination: subsection.4.2 Children: Item: Title: Extraction from Seeds Destination: subsubsection.4.2.1 Item: Title: Enzymatic Characterization of Proteolytic and Inhibitory Activities Destination: subsubsection.4.2.2 Item: Title: Molecular Characterization of Protease Inhibitors Destination: subsubsection.4.2.3 Item: Title: Preparation of ATSI-Trypsin Complex, Crystallization, and Structure Determination Destination: subsubsection.4.2.4 Item: Title: Cell Culture and in Cellulo Inhibitory Assays Destination: subsubsection.4.2.5 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Analysis of Protein Inhibitors of Trypsin in Quinoa, Amaranth and Lupine Seeds. Selection and Deep Structure Function Characterization of the Amaranthus caudatus Species Author: Martha Hernández de la Torre, Giovanni Covaleda-Cortés, Laura Montesinos, Daniela Covaleda, Juan C. Ortiz, Jaume Piñol, José M. Bautista, J. Patricio Castillo, David Reverter and Francesc Xavier Avilés Subject: Protease inhibitors are biomolecules with growing biotechnological and biomedical relevance, including those derived from plants. This study investigated strong trypsin inhibitors in quinoa, amaranth, and lupine seeds, plant grains traditionally used in Andean South America. Amaranth seeds displayed the highest trypsin inhibitory activity, despite having the lowest content of aqueous soluble and thermostable protein material. This activity, directly identified by enzymatic assay, HPLC, intensity-fading mass spectrometry (IF-MS), and MS/MS, was attributed to a single protein of 7889.1 Da, identified as identical in Amaranthus caudatus and A. hybridus, with a Ki of 1.2 nM for the canonical bovine trypsin. This form of the inhibitor, which is highly homogeneous and scalable, was selected, purified, and structurally functionally characterized due to the high nutritional quality of amaranth seeds as well as its promising agriculture biotech biomed applicability. The protein was crystallized in complex with bovine trypsin, and its 3D crystal structure resolved at 2.85 Å, revealing a substrate-like transition state interaction. This verified its classification within the potato I inhibitor family. It also evidenced that the single disulfide bond of the inhibitor constrains its binding loop, which is a key feature. Cell culture assays showed that the inhibitor did not affect the growth of distinct plant microbial pathogen models, including diverse bacteria, fungi, and parasite models, such as Mycoplasma genitalium and Plasmodium falciparum. These findings disfavour the notion that the inhibitor plays an antimicrobial role, favouring its potential as an agricultural insect deterrent and prompting a redirection of its functional research. 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