title: 3D (13)C-(13)C-(13)C correlation NMR for de novo distance determination of solid proteins and application to a human alpha-defensin.
abstract: The de novo structure of an antimicrobial protein, human alpha-defensin 1 (HNP-1), is determined by combining a 3D (13)C-(13)C-(13)C (CCC) magic-angle spinning (MAS) correlation experiment with standard resonance assignment experiments. Using a short spin diffusion mixing time to assign intra-residue cross peaks and a long mixing time to detect inter-residue correlation peaks, we show that the 3D CCC experiment not only reduces the ambiguity of resonance assignment, but more importantly yields two orders of magnitude more long-range distances without recourse to existing crystal structures. Most of these distance constraints could not be obtained in a de novo fashion from 2D correlation spectra due to significant resonance overlap. Combining the distance constraints from the 3D CCC experiment and the chemical-shift-derived torsion angles, we obtained a de novo high-resolution NMR structure of HNP-1, with a heavy-atom RMSD of 3.4A from the crystal structure of the analogous HNP-3. The average energy of the minimum-energy ensemble is less than of 40kcal/mol. Thus, the 3D CCC experiment provides a reliable means of restraining the three-dimensional structure of insoluble proteins with unknown conformations.
authors: Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
authors: Shenhui
authors: S
authors: Li
authors: Yuan
authors: Y
authors: Zhang
authors: Mei
authors: M
authors: Hong
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry*; D003461:Crystallography; D003627:Data Interpretation, Statistical; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D016011:Normal Distribution; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D023084:alpha-Defensins / Q000737:chemistry*
chemical_list: D000890:Anti-Infective Agents; D011506:Proteins; D023084:alpha-Defensins; C406413:human neutrophil peptide 1