title: Single-molecule Force Spectroscopy Predicts a Misfolded, Domain-swapped Conformation in human γD-Crystallin Protein.
abstract: Cataract is a protein misfolding disease where the size of the aggregate is directly related to the severity of the disorder. However, the molecular mechanisms that trigger the onset of aggregation remain unknown. Here we use a combination of protein engineering techniques and single-molecule force spectroscopy using atomic force microscopy to study the individual unfolding pathways of the human γD-crystallin, a multidomain protein that must remain correctly folded during the entire lifetime to guarantee lens transparency. When stretching individual polyproteins containing two neighboring HγD-crystallin monomers, we captured an anomalous misfolded conformation in which the β1 and β2 strands of the N terminus domain of two adjacent monomers swap. This experimentally elusive domain-swapped conformation is likely to be responsible for the increase in molecular aggregation that we measure in vitro. Our results demonstrate the power of force spectroscopy at capturing rare misfolded conformations with potential implications for the understanding of the molecular onset of protein aggregation.
authors.affiliation: From the Department of Physics and Randall Division of Cell and Molecular Biophysics, King's College London, London WC2R 2LS, United Kingdom and sergi.garcia-manyes@kcl.ac.uk.
authors.forename: Sergi
authors.initials: S
authors.lastname: Garcia-Manyes
authors.affiliation: the Department of Biological Sciences, Columbia University, New York, New York 10027.
authors.forename: David
authors.initials: D
authors.lastname: Giganti
authors.affiliation: the Department of Biological Sciences, Columbia University, New York, New York 10027.
authors.forename: Carmen L
authors.initials: CL
authors.lastname: Badilla
authors.affiliation: From the Department of Physics and Randall Division of Cell and Molecular Biophysics, King's College London, London WC2R 2LS, United Kingdom and.
authors.forename: Ainhoa
authors.initials: A
authors.lastname: Lezamiz
authors.affiliation: From the Department of Physics and Randall Division of Cell and Molecular Biophysics, King's College London, London WC2R 2LS, United Kingdom and.
authors.forename: Judit
authors.initials: J
authors.lastname: Perales-Calvo
authors.affiliation: From the Department of Physics and Randall Division of Cell and Molecular Biophysics, King's College London, London WC2R 2LS, United Kingdom and.
authors.forename: Amy E M
authors.initials: AE
authors.lastname: Beedle
authors.affiliation: the Department of Biological Sciences, Columbia University, New York, New York 10027.
authors.forename: Julio M
authors.initials: JM
authors.lastname: Fernández
mesh_terms: D006801:Humans; D066329:Protein Aggregates; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D038222:gamma-Crystallins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C504903:CRYGD protein, human; D066329:Protein Aggregates; D038222:gamma-Crystallins
keywords: atomic force microscopy (AFM); cataract; crystallin; protein misfolding; single-molecule biophysics