title: Transient misfolding dominates multidomain protein folding.
abstract: Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Borgia
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Katherine R
authors.initials: KR
authors.lastname: Kemplen
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Madeleine B
authors.initials: MB
authors.lastname: Borgia
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Andrea
authors.initials: A
authors.lastname: Soranno
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Shammas
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Bengt
authors.initials: B
authors.lastname: Wunderlich
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Nettels
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Best
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Jane
authors.initials: J
authors.lastname: Clarke
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schuler
mesh_terms: D000682:Amyloid; D064211:Connectin / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D044085:Microfluidics; D056004:Molecular Dynamics Simulation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D020449:Repetitive Sequences, Amino Acid
chemical_list: D000682:Amyloid; D064211:Connectin