A novel reverse two-hybrid method for the identification of missense mutations that disrupt protein–protein binding

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dc.contributor.authorVincent, Olivier
dc.contributor.authorGutiérrez Nogués, Ángel
dc.contributor.authorTrejo Herrero, Adrián
dc.contributor.authorNavas Hernández, María Ángeles
dc.date.accessioned2023-12-18T08:26:18Z
dc.date.available2023-12-18T08:26:18Z
dc.date.issued2020-12-03
dc.description.abstractThe reverse two-hybrid system is a powerful method to select mutations that disrupt the interaction between two proteins and therefore to identify the residues involved in this interaction. However, the usefulness of this technique has been limited by its relative complexity when compared to the classical two-hybrid system, since an additional selection step is required to eliminate the high background of uninformative truncation mutants. We have developed a new method that combines the classical and reverse two-hybrid systems to select loss-of-binding missense mutations in a single step. The strategy used to select against truncation mutants is based on the two-hybrid interaction between a C-terminal fusion peptide and the Tsg101 protein. We have applied this method to identify mutations in human glucokinase (GK) that disrupt glucokinase regulatory protein (GKRP) binding. Our results indicate that this method is very efficient and eliminates all the truncation mutants and false positives. The mutated residues identified in GK are involved in the GKRP binding interface or in stabilizing the super-open conformation of GK that binds GKRP. This technique offers an improvement over existing methods in terms of speed, efficiency and simplicity and can be used to study any detectable protein interaction in the two-hybrid system.
dc.description.departmentDepto. de Bioquímica y Biología Molecular
dc.description.facultyFac. de Medicina
dc.description.refereedTRUE
dc.description.sponsorshipInstituto de Salud Carlos III
dc.description.sponsorshipMinisterio de Ciencia e Innovación
dc.description.statuspub
dc.identifier.citationVincent O, Gutierrez-Nogués A, Trejo-Herrero A, Navas MA. A novel reverse two-hybrid method for the identification of missense mutations that disrupt protein-protein binding. Sci Rep. 2020 Dec 3;10(1):21043.
dc.identifier.doi10.1038/s41598-020-77992-1
dc.identifier.issn2045-2322
dc.identifier.relatedurlhttps://www.nature.com/srep/
dc.identifier.urihttps://hdl.handle.net/20.500.14352/91409
dc.journal.titleScientific Reports
dc.language.isoeng
dc.publisherNature Research
dc.relation.projectIDPI100424
dc.relation.projectIDPGC2018- 093604-B-100
dc.rightsAttribution 4.0 Internationalen
dc.rights.accessRightsopen access
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subject.cdu577.2
dc.subject.keywordbiological techniques
dc.subject.keywordmolecular biology
dc.subject.ucmCiencias Biomédicas
dc.subject.ucmCiencias
dc.subject.unesco24 Ciencias de la Vida
dc.titleA novel reverse two-hybrid method for the identification of missense mutations that disrupt protein–protein binding
dc.typejournal article
dc.type.hasVersionVoR
dc.volume.number10
dspace.entity.typePublication
relation.isAuthorOfPublication2996f3da-ebc0-41be-98c4-32a67724a052
relation.isAuthorOfPublication.latestForDiscovery2996f3da-ebc0-41be-98c4-32a67724a052
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