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.
Copyright 2001 Wiley-Liss, Inc.