Suchergebnisse

Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back ∼500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a molecular testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3-cyclopentanedione oxidation, a β–diketone initiator commonly used in vinyl polymerization reactions. ; This study is based upon work funded by the Spanish Government projects BIO2013-43407-R-DEWRY and BIO2016-79106-R-Lignolution and by the Bio Based Industries Joint Undertaking under the European Union's Horizon 2020 Research and Innovation programme (grant agreement no. 886567). B.J.G.-F. was supported by FPI national fellowship BES-2014-068887. ; Peer reviewed

© The Author(s) 2019. ; The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery. ; This work was supported by the European Research Council (grant ERC-2012-ADG-323059 to V.M.) and by the PRODESTECH network funded through the CONSOLIDER program from the Spanish Government (grant CSD2009-00088). L.A.C. acknowledges support from Ministry of Economy and Competitiveness through grants BIO2016-78768-P and RYC-2013-13197. V.M. acknowledges additional support from the W.M. Keck Foundation and from the CREST Center for Cellular and Biomomolecular Machines (grant NSF-CREST-1547848). J.M.V. acknowledges additional support from Ministry of Economy and Competitiveness through grant BFU2016-75984. F.M.R. and A.R. thank the staff from the ALBA synchrotron (Spain) for assistance with the XALOC beamline. Structural data are deposited in the Protein Data Bank with accession codes 6QIY (X-ray CI2 classical geometry) and 6QIZ (X-ray CI2 domain swapped) and EMD-4568 (cryo-EM CI2eng assembly).

The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery. ; This work was supported by the European Research Council (grant ERC-2012-ADG-323059 to V.M.) and by the PRODESTECH network funded through the CONSOLIDER program from the Spanish Government (grant CSD2009-00088). L.A.C. acknowledges support from Ministry of Economy and Competitiveness through grants BIO2016-78768-P and RYC-2013-13197. V.M. acknowledges additional support from the W.M.Keck Foundation and from the CREST Center for Cellular and Biomomolecular Machines (grant NSF-CREST-1547848). J.M.V. acknowledges additional support from Ministry of Economy and Competitiveness through grant BFU2016-75984. ; Peer reviewed