title: EPR mapping of interactions between spin-labeled variants of human carbonic anhydrase II and GroEL: evidence for increased flexibility of the hydrophobic core by the interaction.
abstract: Human carbonic anhydrase II (HCA II) interacts weakly with GroEL at room temperature. To further investigate this interaction we used electron paramagnetic resonance (EPR) spectroscopy to study HCA II cysteine mutants spin-labeled at selected positions. From our results it is evident that protein-protein interactions can be specifically mapped by site-directed spin-labeling and EPR measurements. HCA II needs to be unfolded to about the same extent as a GuHCl-induced molten-globule intermediate of the enzyme to interact with GroEL. The interaction with GroEL includes interactions with outer parts of the HCA II molecule, such as peripheral beta-strands and the N-terminal domain, which have previously been shown to be rather unstable. As a result of the interaction, the rigid and compact hydrophobic core exhibits higher flexibility than in the molten globule, which is likely to facilitate rearrangements of misfolded structure during the folding process. The degree of binding to GroEL and accompanying inactivation of the enzyme depend on the stability of the HCA II variant, and nonspecific hydrophobic interactions appear to be most important in stabilizing the GroEL-substrate complex.
authors.affiliation: IFM-Department of Chemistry, Linköping University, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Persson
authors.forename: P
authors.initials: P
authors.lastname: Hammarström
authors.forename: M
authors.initials: M
authors.lastname: Lindgren
authors.forename: B H
authors.initials: BH
authors.lastname: Jonsson
authors.forename: M
authors.initials: M
authors.lastname: Svensson
authors.forename: U
authors.initials: U
authors.lastname: Carlsson
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry*; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010449:Peptide Mapping; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013113:Spin Labels; D014364:Tryptophan / Q000235:genetics
chemical_list: D018834:Chaperonin 60; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013113:Spin Labels; D010649:Phenylalanine; D014364:Tryptophan; D002256:Carbonic Anhydrases; D003545:Cysteine