title: Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three-Stranded Coiled-Coil Structure Containing d-Amino Acids.
abstract: The core objective of de novo metalloprotein design is to define metal-protein relationships that control the structure and function of metal centers by using simplified proteins. An essential requirement to achieve this goal is to obtain high resolution structural data using either NMR or crystallographic studies in order to evaluate successful design. X-ray crystal structures have proven that a four heptad repeat scaffold contained in the three-stranded coiled coil (3SCC), called CoilSer (CS), provides an excellent motif for modeling a three Cys binding environment capable of chelating metals into geometries that resemble heavy metal sites in metalloregulatory systems. However, new generations of more complicated designs that feature, for example, a d-amino acid or multiple metal ligand sites in the helical sequence require a more stable construct. In doing so, an extra heptad was introduced into the original CS sequence, yielding a GRAND-CoilSer (GRAND-CS) to retain the 3SCC folding. An apo-(GRAND-CSL12DLL16C)3 crystal structure, designed for Cd(II)S3 complexation, proved to be a well-folded parallel 3SCC. Because this structure is novel, protocols for crystallization, structural determination, and refinements of the apo-(GRAND-CSL12DLL16C)3 are described. This report should be generally useful for future crystallographic studies of related coiled-coil designs.
authors: Department of Chemistry, University of Michigan, Ann Arbor, MI, United States.
authors: L
authors: L
authors: Ruckthong
authors: Life Sciences Institute, University of Michigan, Ann Arbor, MI, United States.
authors: J A
authors: JA
authors: Stuckey
authors: Department of Chemistry, University of Michigan, Ann Arbor, MI, United States. Electronic address: vlpec@umich.edu.
authors: V L
authors: VL
authors: Pecoraro
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008024:Ligands; D008667:Metalloproteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D008670:Metals; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D017433:Protein Structure, Secondary
chemical_list: D000596:Amino Acids; D008024:Ligands; D008667:Metalloproteins; D008670:Metals; D010455:Peptides
keywords: De novo protein design; Three-stranded coiled coil; X-ray crystallography; d-Amino acid