FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3083353607> ?p ?o ?g. }

• W3083353607 abstract "Coagulation Factor XII (FXII) is an important protein involved in the initiation of the intrinsic pathway of the coagulation cascade. Recent studies suggested that FXII may play a role in pathological thrombosis without compromising physiological hemostasis. For this reason, the inhibition of FXII could represent a new and selective strategy for preventing stroke and other thromboembolic diseases. Since there is no structure available for the protease domain of FXII, the knowledge of the overall domain structure can be significantly helpful in the development of inhibitors with specific selectivity for the target.Three different constructs were made: βFXIIa, βFXIIG570R, a missense mutations that causes a reduced activity of FXIIa, and βFXIIa fused with Maltose Binding Protein (MBP) at the N-terminus. An expression and purification protocol was established for all the proteins. In order to assess the activity of the recombinant protein, the hydrolysis of chromogenic substrate S2302 was measured and a comparison with commercial αFXIIa and βFXIIa was performed. The kinetic parameters (kcat, KM, kcat/KM, Vmax) indicated that the catalytic behaviour of recombinant MBP-βFXIIa and βFXIIa is essentially identical to that of commercial βFXIIa purified from plasma. Therefore, the protein expressed and purified from insect cell system is catalytically competent.Large efforts were devoted to the identification of possible crystallization conditions for the expressed and purified constructs, in complex with different inhibitors, both small molecules and macromolecular inhibitors. Diffracting crystals were obtained for MBP-βFXIIa in complex with D-Phe-Pro-Argchloromethylketone (PPACK). A complete data set was collected at 4 A. Notwithstanding the low resolution of the data, the structural analysis of key elements and the comparison with the zymogen confirmed that recombinant protein is in the active conformation. Moreover, a first analysis of the structure and of the lattice packing enabled to understand some key differences between the interactions that PPACK forms when in complex to FXIIa and with thrombin, thus possible explaining the lower affinity of PPACK for FXIIa. This is the first crystallographic structure of FXIIa and it represents a first step in the study of protein-inhibitor interactions from structural consideration.βFXII construct was also cloned in a different vector for the expression in E. coli. The cleavage of TF tag was performed using three different enzymes (HRV-3C protease, FXa and thrombin). The best results were obtained, using the thrombin and setting the reaction overnight at room temperature. However, it was not possible to continue the process, since the protein was lost after the cleavage. This could be due to a poor stability of the protein. For that reason, a different strategy was developed, consisting of co-purification of βFXII with Ecotin. The presence of the inhibitor was thought to stabilize the protein during the cleavage. However, the gel filtration revealed that the complex was not formed.The possibility to investigate at atomic level the interactions of the inhibitors with FXII could allow an understanding as to how the substrate-inhibitor recognition mechanism work. This could therefore represent a good starting point for the development of new inhibitors that have higher specificity for FXII." @default.
• W3083353607 created "2020-09-11" @default.
• W3083353607 creator A5005571955 @default.
• W3083353607 date "2016-01-01" @default.
• W3083353607 modified "2023-09-26" @default.
• W3083353607 title "Crystallographic studies of coagulation factor XII" @default.
• W3083353607 hasPublicationYear "2016" @default.
• W3083353607 type Work @default.
• W3083353607 sameAs 3083353607 @default.
• W3083353607 citedByCount "0" @default.
• W3083353607 crossrefType "dissertation" @default.
• W3083353607 hasAuthorship W3083353607A5005571955 @default.
• W3083353607 hasConcept C118552586 @default.
• W3083353607 hasConcept C181199279 @default.
• W3083353607 hasConcept C185592680 @default.
• W3083353607 hasConcept C2777167447 @default.
• W3083353607 hasConcept C2778382381 @default.
• W3083353607 hasConcept C41183919 @default.
• W3083353607 hasConcept C52853521 @default.
• W3083353607 hasConcept C55493867 @default.
• W3083353607 hasConcept C56856141 @default.
• W3083353607 hasConcept C71924100 @default.
• W3083353607 hasConceptScore W3083353607C118552586 @default.
• W3083353607 hasConceptScore W3083353607C181199279 @default.
• W3083353607 hasConceptScore W3083353607C185592680 @default.
• W3083353607 hasConceptScore W3083353607C2777167447 @default.
• W3083353607 hasConceptScore W3083353607C2778382381 @default.
• W3083353607 hasConceptScore W3083353607C41183919 @default.
• W3083353607 hasConceptScore W3083353607C52853521 @default.
• W3083353607 hasConceptScore W3083353607C55493867 @default.
• W3083353607 hasConceptScore W3083353607C56856141 @default.
• W3083353607 hasConceptScore W3083353607C71924100 @default.
• W3083353607 hasLocation W30833536071 @default.
• W3083353607 hasOpenAccess W3083353607 @default.
• W3083353607 hasPrimaryLocation W30833536071 @default.
• W3083353607 hasRelatedWork W1826771983 @default.
• W3083353607 hasRelatedWork W1973642951 @default.
• W3083353607 hasRelatedWork W2030636773 @default.
• W3083353607 hasRelatedWork W2057179438 @default.
• W3083353607 hasRelatedWork W2077898624 @default.
• W3083353607 hasRelatedWork W2085892916 @default.
• W3083353607 hasRelatedWork W2093067825 @default.
• W3083353607 hasRelatedWork W2157506648 @default.
• W3083353607 hasRelatedWork W2268388363 @default.
• W3083353607 hasRelatedWork W2535197426 @default.
• W3083353607 hasRelatedWork W2565116432 @default.
• W3083353607 hasRelatedWork W2569964808 @default.
• W3083353607 hasRelatedWork W2580462625 @default.
• W3083353607 hasRelatedWork W2900851732 @default.
• W3083353607 hasRelatedWork W2910886109 @default.
• W3083353607 hasRelatedWork W2925014580 @default.
• W3083353607 hasRelatedWork W2948028981 @default.
• W3083353607 hasRelatedWork W2998912706 @default.
• W3083353607 hasRelatedWork W3093870597 @default.
• W3083353607 hasRelatedWork W41252578 @default.
• W3083353607 isParatext "false" @default.
• W3083353607 isRetracted "false" @default.
• W3083353607 magId "3083353607" @default.
• W3083353607 workType "dissertation" @default.