title: Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering, Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation.
abstract: The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution.
authors.affiliation: Department of Chemistry-Ångström laboratory, Uppsala University, Box 523, SE-75110 Uppsala, Sweden.
authors.forename: Alexandr
authors.initials: A
authors.lastname: Nasedkin
authors.affiliation: Uppsala Center for Computational Chemistry, Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, Box 596, SE-75124 Uppsala, Sweden.
authors.forename: Moreno
authors.initials: M
authors.lastname: Marcellini
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, United States.
authors.forename: Tomasz L
authors.initials: TL
authors.lastname: Religa
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
authors.forename: Stefan M
authors.initials: SM
authors.lastname: Freund
authors.affiliation: Paul Scherrer Institut, 5232 Villigen-PSI, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Menzel
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
authors.affiliation: Uppsala Center for Computational Chemistry, Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, Box 596, SE-75124 Uppsala, Sweden.
authors.forename: David
authors.initials: D
authors.lastname: van der Spoel
authors.affiliation: Department of Chemistry-Ångström laboratory, Uppsala University, Box 523, SE-75110 Uppsala, Sweden.
authors.forename: Jan
authors.initials: J
authors.lastname: Davidsson
mesh_terms: D000818:Animals; D016000:Cluster Analysis; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000032:analysis / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism*; D014965:X-Rays
chemical_list: D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D014157:Transcription Factors