title: Design and initial characterization of a circular permuted variant of the potent HIV-inactivating protein cyanovirin-N.
abstract: A circular permuted variant of the potent human immunodeficiency virus (HIV)-inactivating protein cyanovirin-N (CV-N) was constructed. New N- and C-termini were introduced into an exposed helical loop, and the original termini were linked using residues of the original loop. Since the three-dimensional structure of wild-type cyanovirin-N is a pseudodimer, the mutant essentially exhibits a swap between the two pseudo-symmetrically related halves. The expressed protein, which accumulates in the insoluble fraction, was purified, and conditions for in vitro refolding were established. During refolding, a transient dimeric species is also formed that converts to a monomer. Similar to the wild-type CV-N, the monomeric circular permuted protein exhibits reversible thermal unfolding and urea denaturation. The mutant is moderately less stable than the wild-type protein, but it displays significantly reduced anti-HIV activity. Using nuclear magnetic resonance spectroscopy, we demonstrate that this circular permuted monomeric molecule adopts the same fold as the wild-type protein. Characterization of these two architecturally very similar molecules allows us to embark, for the first time, on a structure guided focused mutational study, aimed at delineating crucial features for the extraordinary difference in the activity of these molecules.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
authors.forename: Laura G
authors.initials: LG
authors.lastname: Barrientos
authors.forename: John M
authors.initials: JM
authors.lastname: Louis
authors.forename: Jason
authors.initials: J
authors.lastname: Hung
authors.forename: Trent H
authors.initials: TH
authors.lastname: Smith
authors.forename: Barry R
authors.initials: BR
authors.lastname: O'Keefe
authors.forename: Roberta S
authors.initials: RS
authors.lastname: Gardella
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Mori
authors.forename: Michael R
authors.initials: MR
authors.lastname: Boyd
authors.forename: Angela M
authors.initials: AM
authors.lastname: Gronenborn
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D019281:Dimerization; D015195:Drug Design; D004926:Escherichia coli; D015497:HIV-1 / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding
chemical_list: D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; C107733:cyanovirin N