title: The importance of being knotted: effects of the C-terminal knot structure on enzymatic and mechanical properties of bovine carbonic anhydrase II.
abstract: In order to better understand the contribution of the knotted folding pattern to the enzymatic and mechanical properties of carbonic anhydrases, we replaced Gln-253 of bovine carbonic anhydrase II with Cys, which allowed us to measure the mechanical strength of the protein against tensile deformation by avoiding knot tightening. The expressed protein, to our surprise, turned out to contain two conformational isomers, one capable of binding an enzymatic inhibitor and the other not, which led to their separation through affinity chromatography. In near- and far-UV circular dichroism and fluorescence spectra, the separated conformers were very similar to each other and to the wild-type enzyme, indicating that they both had native-like conformations. We describe new evidence which supports the notion that the difference between the two conformers is likely to be related to the completeness of the C-terminal knot formation.
authors.affiliation: Laboratory of Biodynamics, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan.
authors.forename: Mohammad Taufiq
authors.initials: MT
authors.lastname: Alam
authors.forename: Takafumi
authors.initials: T
authors.lastname: Yamada
authors.forename: Uno
authors.initials: U
authors.lastname: Carlsson
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ikai
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D018833:Chaperonins / Q000737:chemistry; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000502:physiology; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D020673:Protein Renaturation; D013050:Spectrometry, Fluorescence; D013314:Stress, Mechanical
chemical_list: D018833:Chaperonins; D024402:Carbonic Anhydrase II