title: Thermodynamics of a designed protein catenane.
abstract: Topological linking of proteins is a new approach for stabilizing and controlling the oligomerization state of proteins that fold in an interwined manner. The recent design of a backbone cyclized protein catenane based on the p53tet domain suggested that topological cross-linking provided increased stability against thermal and chemical denaturation. However, the tetrameric structure complicated detailed biophysical analysis of this protein. Here, we describe the design, synthesis and thermodynamic characterization of a protein catenane based on a dimeric mutant of the p53tet domain (M340E/L344K). The formation of the catenane proceeded efficiently, and the overall structure and oligomerization of the domain was not affected by the formation of the topological link. Unfolding and refolding of the catenane was consistent with a two-state process. The topological link stabilized the dimer against thermal and chemical denaturation considerably, raising the apparent melting temperature by 59 degrees C and the midpoint of denaturation by 4.5M GuHCl at a concentration of 50 microM. The formation of the topological link increased the resistance of the dimer to proteolysis. However, the m value decreased by 1.7kcalmol(-1)M(-1), suggesting a decrease in accessible surface area in the unfolded state. This implies that the stabilization from the topological link is largely due to a destabilization of the unfolded state, similar to other cross-links in proteins. Topological linking therefore provides a powerful and orthogonal tool for the stabilization of peptide and protein oligomers.
authors.affiliation: Department of Cell Biology and Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MC CVN-6, La Jolla, CA 92037, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Blankenship
authors.forename: Philip E
authors.initials: PE
authors.lastname: Dawson
mesh_terms: D000873:Anthracenes / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D019791:Guanidine; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D010447:Peptide Hydrolases / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D000873:Anthracenes; D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D011083:Polycyclic Compounds; D016159:Tumor Suppressor Protein p53; C467767:catenane; D010447:Peptide Hydrolases; D019791:Guanidine