title: Mapping the Topography of a Protein Energy Landscape.
abstract: Protein energy landscapes are highly complex, yet the vast majority of states within them tend to be invisible to experimentalists. Here, using site-directed mutagenesis and exploiting the simplicity of tandem-repeat protein structures, we delineate a network of these states and the routes between them. We show that our target, gankyrin, a 226-residue 7-ankyrin-repeat protein, can access two alternative (un)folding pathways. We resolve intermediates as well as transition states, constituting a comprehensive series of snapshots that map early and late stages of the two pathways and show both to be polarized such that the repeat array progressively unravels from one end of the molecule or the other. Strikingly, we find that the protein folds via one pathway but unfolds via a different one. The origins of this behavior can be rationalized using the numerical results of a simple statistical mechanics model that allows us to visualize the equilibrium behavior as well as single-molecule folding/unfolding trajectories, thereby filling in the gaps that are not accessible to direct experimental observation. Our study highlights the complexity of repeat-protein folding arising from their symmetrical structures; at the same time, however, this structural simplicity enables us to dissect the complexity and thereby map the precise topography of the energy landscape in full breadth and remarkable detail. That we can recapitulate the key features of the folding mechanism by computational analysis of the native structure alone will help toward the ultimate goal of designed amino-acid sequences with made-to-measure folding mechanisms-the Holy Grail of protein folding.
authors.affiliation: Hutchison/MRC Research Centre , Hills Road, Cambridge CB2 0XZ, U.K.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Hutton
authors.affiliation: Hutchison/MRC Research Centre , Hills Road, Cambridge CB2 0XZ, U.K.
authors.forename: James
authors.initials: J
authors.lastname: Wilkinson
authors.affiliation: ICTEAM, Université Catholique de Lovain , Euler Building 4, Avenue Lemaître, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Mauro
authors.initials: M
authors.lastname: Faccin
authors.affiliation: Department of Pharmacology, University of Cambridge , Tennis Court Road, Cambridge CB2 1PD, U.K.
authors.forename: Elin M
authors.initials: EM
authors.lastname: Sivertsson
authors.affiliation: Dipartimento di Scienza Applicata e Tecnologia, CNISM, and Center for Computational Studies, Politecnico di Torino , Corso Duca degli Abruzzi 24, I-10129 Torino, Italy.|INFN, Sezione di Torino , via Pietro Giuria 1, I-10125 Torino, Italy.|Human Genetics Foundation (HuGeF) , Via Nizza 52, I-10126 Torino, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pelizzola
authors.affiliation: Institute of Structural and Molecular Biology and London Centre for Nanotechnology, University College London and Birkbeck College , London WC1E 7HX, U.K.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Lowe
authors.affiliation: Departamento de Física Teórica and Instituto de Biocomputacíon y Física de Sistemas Complejos (BIFI), Universidad de Zaragoza , c/Mariano Esquillor s/n, 50018 Zaragoza, Spain.
authors.forename: Pierpaolo
authors.initials: P
authors.lastname: Bruscolini
authors.affiliation: Department of Pharmacology, University of Cambridge , Tennis Court Road, Cambridge CB2 1PD, U.K.
authors.forename: Laura S
authors.initials: LS
authors.lastname: Itzhaki
mesh_terms: D007700:Kinetics; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins