title: Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates.
abstract: Cyclophilin catalyzes the ubiquitous process "peptidyl-prolyl cis-trans isomerization," which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by1H-15N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.
authors.affiliation: UM-DAE-Centre for Excellence in Basic Sciences, University of Mumbai, Kalina Campus, Mumbai, India.
authors.forename: Vaibhav Kumar
authors.initials: VK
authors.lastname: Shukla
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology, Mumbai, India.
authors.forename: Jai Shankar
authors.initials: JS
authors.lastname: Singh
authors.affiliation: UM-DAE-Centre for Excellence in Basic Sciences, University of Mumbai, Kalina Campus, Mumbai, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Vispute
authors.affiliation: UM-DAE-Centre for Excellence in Basic Sciences, University of Mumbai, Kalina Campus, Mumbai, India.
authors.forename: Basir
authors.initials: B
authors.lastname: Ahmad
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology, Mumbai, India. Electronic address: ashutoshk@iitb.ac.in.
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Kumar
authors.affiliation: UM-DAE-Centre for Excellence in Basic Sciences, University of Mumbai, Kalina Campus, Mumbai, India; Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India. Electronic address: hosur@tifr.res.in.
authors.forename: Ramakrishna V
authors.initials: RV
authors.lastname: Hosur
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D021983:Cyclophilins / Q000737:chemistry*; D008958:Models, Molecular; D000072756:Protein Conformation, alpha-Helical / Q000187:drug effects; D000072757:Protein Conformation, beta-Strand / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology
chemical_list: D029702:Schizosaccharomyces pombe Proteins; D014508:Urea; D021983:Cyclophilins; C479571:cyp3 protein, S pombe