title: Characterization of folding intermediates of a domain-swapped protein by solid-state NMR spectroscopy.
abstract: We have employed two-dimensional solid-state NMR to study structure and dynamics of insoluble folding states of the domain-swapped protein Crh. Starting from the protein precipitated at its pI, conformational changes due to a modest temperature increase were investigated at the level of individual residues and in real-time. As compared to the crystalline state, Crh pI-precipitates exhibited a higher degree of molecular mobility for several regions of the protein. A rigidly intact center was observed including a subset of residues of the hydrophobic core. Raising the temperature by 13 K to 282 K created a partially unfolded intermediate state that was converted into beta-sheet-rich aggregates that are mostly of spherical character according to electron microscopy. Residue-by-residue analysis indicated that two out of three alpha-helices in aggregated Crh underwent major structural rearrangements while the third helix was preserved. Residues in the hinge region exhibited major chemical-shift changes, indicating that the domain swap was not conserved in the aggregated form. Our study provides direct evidence that protein aggregates of a domain-swapped protein retain a significant fraction of native secondary structure and demonstrates that solid-state NMR can be used to directly monitor slow molecular folding events.
authors.affiliation: Department of NMR-Based Structural Biology and Electron Microscopy, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Etzkorn
authors.forename: Anja
authors.initials: A
authors.lastname: Böckmann
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.forename: Dietmar
authors.initials: D
authors.lastname: Riedel
authors.forename: Marc
authors.initials: M
authors.lastname: Baldus
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008854:Microscopy, Electron; D010750:Phosphoproteins / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D001426:Bacterial Proteins; C118042:Crh protein, Bacillus subtilis; D010750:Phosphoproteins