Design and initial characterization of a circular permuted variant of the potent HIV-inactivating protein cyanovirin-N.
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.
Barrientos
Laura G
LG
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Louis
John M
JM
Hung
Jason
J
Smith
Trent H
TH
O'Keefe
Barry R
BR
Gardella
Roberta S
RS
Mori
Toshiyuki
T
Boyd
Michael R
MR
Gronenborn
Angela M
AM
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
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; C107733:cyanovirin N