title: Conversion of two-state to multi-state folding kinetics on fusion of two protein foldons.
abstract: Chymotrypsin inhibitor 2 (CI2) is the archetypal single-foldon protein that folds in simple two-state kinetics without the accumulation of a folding intermediate. To model the effects of fusion of single foldons to give a multi-foldon protein, we engineered a "double-CI2" protein, in which another CI2 polypeptide was inserted into the loop region of the parent CI2. CD and HSQC spectra demonstrated that while the double-CI2 protein adopted two kinds of native conformations, CI2-like structure was almost preserved in both the domains of double-CI2. In the folding kinetic studies, double-CI2 exhibited a remarkable rollover of the observed folding rates at low denaturant concentrations, indicating that double-CI2 accumulated a kinetic folding intermediate. The different folding mechanisms between WT-CI2 and double-CI2 support the present view that protein size or number of domains is an important determinant for formation of folding intermediates.
authors.affiliation: Cambridge Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge, CB2 2QH, UK.
authors.forename: K
authors.initials: K
authors.lastname: Inaba
authors.forename: N
authors.initials: N
authors.lastname: Kobayashi
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins; D011489:Protein Denaturation / Q000187:drug effects; D015202:Protein Engineering; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D014466:Ultraviolet Rays
chemical_list: D010455:Peptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C093336:chymotrypsin inhibitor 2; D019791:Guanidine