Characterization of folding intermediates of a domain-swapped protein by solid-state NMR spectroscopy.
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.
Etzkorn
Manuel
M
Department of NMR-Based Structural Biology and Electron Microscopy, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Böckmann
Anja
A
Penin
François
F
Riedel
Dietmar
D
Baldus
Marc
M
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
D001426:Bacterial Proteins; C118042:Crh protein, Bacillus subtilis; D010750:Phosphoproteins