{"id":10357,"date":"2022-01-01T11:30:00","date_gmt":"2022-01-01T10:30:00","guid":{"rendered":"https:\/\/www.toulouse-biotechnology-institute.fr\/?p=10357"},"modified":"2022-07-21T11:32:17","modified_gmt":"2022-07-21T09:32:17","slug":"design-de-proteines-mutlidomaines-etudier-la-geometrie-spatiale-entre-enzymes-ou-fonctionaliser-la-cellulose-deux-approches-possibles","status":"publish","type":"post","link":"https:\/\/www.toulouse-biotechnology-institute.fr\/en\/design-de-proteines-mutlidomaines-etudier-la-geometrie-spatiale-entre-enzymes-ou-fonctionaliser-la-cellulose-deux-approches-possibles\/","title":{"rendered":"Design de proteines mutlidomaines; \u00e9tudier la g\u00e9om\u00e9trie spatiale entre enzymes ou fonctionaliser la cellulose, deux approches possibles."},"content":{"rendered":"
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Ind\u00e9pendamment de leur origine biologique, la plupart des prot\u00e9ines sont compos\u00e9es de plusieurs domaines \u00e9l\u00e9mentaires reli\u00e9s entre eux par des connecteurs prot\u00e9iques. Ces domaines sont soit des unit\u00e9s fonctionnellement ind\u00e9pendantes, soit des parties de structures multidomaines plus larges, dont les fonctions sont li\u00e9es \u00e0 leur proximit\u00e9 spatiale. C\u2019est le cas pour les enzymes impliqu\u00e9es dans la d\u00e9construction des parois v\u00e9g\u00e9tales. Afin d\u2019interroger l\u2019effet de la proximit\u00e9 et de la g\u00e9om\u00e9trie spatiale de ces domaines sur la catalyse, nous avons utilis\u00e9 une approche originale qui consiste \u00e0 souder entre eux deux domaines en utilisant deux petites prot\u00e9ines Jo et In ; la particularit\u00e9 est que Jo et In nous permettent de figer nos enzymes dans des conformations spatiales contr\u00f4l\u00e9es et de tester leur activit\u00e9 sur des substrats insolubles.<\/p>

Une autre approche a consist\u00e9 \u00e0 cr\u00e9er une plateforme prot\u00e9ique chim\u00e9rique constitu\u00e9e de trois domaines, dont un permet une association avec la cellulose, tandis que les deux autres domaines permettent d\u2019y associer une large gamme de mol\u00e9cules chimique ou biologique. Le but est de pouvoir fonctionnaliser la cellulose pour la valoriser. C\u2019est l\u2019ensemble de ces approches de design de prot\u00e9ines mutlidomaines que nous pr\u00e9sentons ici.<\/p>

Dans l\u2019optique d\u2019une neutralit\u00e9 carbone \u00e0 l\u2019horizon 2050, la valorisation de la biomasse v\u00e9g\u00e9tale comme source de carbone renouvelable pour la production d\u2019\u00e9nergie, de mat\u00e9riaux bio-sourc\u00e9s ou de mol\u00e9cules chimiques fait partie de l\u2019\u00e9quation. Les microorganismes lignocellulolytiques ont d\u00e9velopp\u00e9s tout un arsenal d\u2019enzymes leur permettant de d\u00e9construire la biomasse v\u00e9g\u00e9tale et de m\u00e9taboliser le carbone contenu dans les polysaccharides des parois cellulaires. Ces enzymes sont souvent multidomaines et interagissent in vivo<\/em> avec des substrats insolubles complexes enchev\u00eatr\u00e9s les uns aux autres. Alors que les domaines de ces enzymes sont en g\u00e9n\u00e9ral bien caract\u00e9ris\u00e9s in vitro<\/em> sur des substrats synth\u00e9tiques solubles, ils le sont rarement dans leur environnement naturel, dense et complexe. Il semble donc pertinent d\u2019interroger l\u2019activit\u00e9 de ces enzymes multidomaines dans leur globalit\u00e9 tout en prenant en compte la conformation et la g\u00e9om\u00e9trie spatiale que ces diff\u00e9rents domaines peuvent adopter au cours de la catalyse et comment celles-ci influent sur les produits de la catalyse. Mais cette multimodularit\u00e9 peut aussi \u00eatre mise \u00e0 profit pour valoriser certaine fraction de la paroi, comme la cellulose. Ainsi, ce polym\u00e8re abondant est d\u00e9j\u00e0 utilis\u00e9 dans de nombreux secteurs industriels. Faciliter et \u00e9tendre la possibilit\u00e9 de fonctionnaliser ce biomat\u00e9riau permettraient d\u2019accroitre ses applications.<\/p>

Jo et In sont deux prot\u00e9ines qui s\u2019associent de mani\u00e8re covalente et spontan\u00e9e. L\u2019asym\u00e9trie de cette association ainsi que leur position dans une prot\u00e9ine multidomaine nous a permis de contr\u00f4ler la g\u00e9om\u00e9trie spatiale entre deux domaines; une enzyme d\u00e9gradant un polysaccharide de la paroi v\u00e9g\u00e9tale et un domaine de reconnaissance de sucres (CBM). Nous avons r\u00e9solue les mod\u00e8les de ces prot\u00e9ines par Small angle X-ray scattering (SAXS) afin d\u2019avoir une image en solution de la position et de la distance entre les deux domaines. Puis nous avons \u00e9tudi\u00e9 l\u2019activit\u00e9 biologique des diff\u00e9rents domaines sur des substrats simples et solubles, jusqu\u2019\u00e0 des substrats naturels et insolubles, comme la paille ou le son de bl\u00e9. Les r\u00e9sultats montrent que nos enzymes multidomaines pr\u00e9sentent bien une g\u00e9om\u00e9trie spatiale d\u00e9pendante de la mani\u00e8re dont les domaines sont assembl\u00e9s. La rigidit\u00e9 de l\u2019ensemble due au complexe Jo-In emp\u00eache toute interf\u00e9rence physique entre les domaines. De plus, en fonction de la position de Jo et de In, on peut jouer sur l\u2019angle de torsion form\u00e9 entre les deux domaines. Nous avons montr\u00e9 que cette diff\u00e9rence de topologie entre enzyme et CBM avait un impact sur l\u2019activit\u00e9 enzymatique dans des parois de cellules v\u00e9g\u00e9tales.<\/p>

Nous avons \u00e9galement d\u00e9velopp\u00e9 une plateforme mutlidomaine centr\u00e9e sur une prot\u00e9ine pr\u00e9sentant une forte affinit\u00e9 pour la cellulose. De part et d\u2019autre de cette prot\u00e9ine, nous avons fusionn\u00e9 de mani\u00e8re plus classique deux autres domaines prot\u00e9iques; l\u2019un permet d\u2019associer des mol\u00e9cules ou des prot\u00e9ines biotynil\u00e9es, l\u2019autre d\u2019associer par click chimie \u00e0 l\u2019aide de l\u2019incorporation d\u2019un acide amin\u00e9 non naturel dans la s\u00e9quence prot\u00e9ique des mol\u00e9cules chimiques. Il est ainsi possible d\u2019apporter \u00e0 la cellulose au moins deux fonctions d\u2019int\u00e9r\u00eat. La mod\u00e9lisation mol\u00e9culaire de notre prot\u00e9ine chim\u00e9rique nous a permis d\u2019appr\u00e9hender la flexibilit\u00e9 de notre mod\u00e8le et de valider son utilisation. Ainsi, \u00e0 l\u2019aide d\u2019une seule prot\u00e9ine multidomaine, nous avons marqu\u00e9 par fluorescence des nanocristaux de cellulose que nous avons immobilis\u00e9 sur des billes paramagn\u00e9tiques pour les isoler.<\/p>

R\u00e9f\u00e9rences bibliographiques<\/strong> :<\/p>

  1. Bonnet, J. Cartannaz, G. Tourcier, C. Contreras-Martel, J. P. Kleman, C. Morlot, T. Vernet and A. M. Di Guilmi, Sci. Rep., 2017, 7, 43564.<\/li>
  2. Enjalbert, M. De La Mare, P. Roblin, L. Badruna, T. Vernet, C. Dumon and C. Y. Montanier, Int. J. Mol. Sci., 2020, 21, 4360.<\/li>
  3. Y. Montanier, M. Fanuel, H. Rogniaux, D. Ropartz, A.-M. Di Guilmi and A. Bouchoux, Sci. Rep., 2019, 9, 5763.<\/li><\/ol>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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    Contact : <\/strong>cedric.montanier@insa-toulouse.fr<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<\/div>","protected":false},"excerpt":{"rendered":"

    Ind\u00e9pendamment de leur origine biologique, la plupart des prot\u00e9ines sont compos\u00e9es de plusieurs domaines \u00e9l\u00e9mentaires reli\u00e9s entre eux par des connecteurs prot\u00e9iques. Ces domaines sont soit des unit\u00e9s fonctionnellement ind\u00e9pendantes, soit des parties de structures multidomaines plus larges, dont les fonctions sont li\u00e9es \u00e0 leur proximit\u00e9 spatiale. C\u2019est le cas pour les enzymes impliqu\u00e9es dans […]<\/p>\n","protected":false},"author":1,"featured_media":10358,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[18],"tags":[35],"class_list":["post-10357","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-actus","tag-equipe-cimes"],"yoast_head":"\nDesign de proteines mutlidomaines; \u00e9tudier la g\u00e9om\u00e9trie spatiale entre enzymes ou fonctionaliser la cellulose, deux approches possibles. | TBI, Toulouse Biotechnology Institute<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.toulouse-biotechnology-institute.fr\/en\/design-de-proteines-mutlidomaines-etudier-la-geometrie-spatiale-entre-enzymes-ou-fonctionaliser-la-cellulose-deux-approches-possibles\/\" \/>\n<meta property=\"og:locale\" content=\"en_GB\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Design de proteines mutlidomaines; \u00e9tudier la g\u00e9om\u00e9trie spatiale entre enzymes ou fonctionaliser la cellulose, deux approches possibles. | TBI, Toulouse Biotechnology Institute\" \/>\n<meta property=\"og:description\" content=\"Ind\u00e9pendamment de leur origine biologique, la plupart des prot\u00e9ines sont compos\u00e9es de plusieurs domaines \u00e9l\u00e9mentaires reli\u00e9s entre eux par des connecteurs prot\u00e9iques. 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