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.

Rational and Computational Design of Stabilized Variants of Cyanovirin-N That Retain Affinity and Specificity for Glycan Ligands

“…The proteins possess identical secondary and tertiary structure, as judged by far- and near-ultraviolet CD spectroscopy (Figure 3). The far-UV CD spectra of the variants were similar to previously-published spectra for wild-type CVN (70). In addition, the intrinsic tryptophan fluorescence emission for each variant was blue-shifted 20 nm compared to the fluorescence of GuaHCl-denatured protein (not shown), indicating the burial of the unique Trp in CVN (Trp49).…”

“…In addition, other biophysical studies used CVN variants with an additional N-terminal Gly–Ser–His–Met–Gly sequence which remained after thrombin cleavage. CVN containing these additional five animo acids at the N-terminus exhibited anti-HIV activity which is indistinguishable from wild-type protein (10, 69, 70), yet may have thermodynamic properties different from wild type.…”

Designed oligomers of cyanovirin-N show enhanced HIV neutralization

“…The harvested cells were lysed using an EmulsiFlex-C5 (Avestin, Inc.), and the insoluble fraction was resuspended in buffer containing 6 M GnHCl and 10 mM imidazole and centrifuged to remove debris. The solubilized CV-N was then purified under denaturing conditions using a Ni-NTA gravity column (Qiagen) and refolded by dialyzing the Ni-NTA eluate against native buffer overnight at room temperature (42). Following refolding, proteins were additionally purified on a Superdex-75 column and eluted in 25 mM sodium phosphate pH 7.4, 150 mM NaCl.…”

Cyanovirin-N: a sugar-binding antiviral protein with a new twist

“…Using residual dipolar couplings, it was shown that dsCV-N has the same overall shape and possibly structure as wild-type CV-N [38]. This construct can also be described as a variation of a circularly-permuted form of CV-N (cpCV-N) in which several residues are shifted in their sequence [12,39]. The monomeric cpCV-N was shown to be moderately less stable than the wild-type CV-N and to display significantly reduced anti-HIV activity.…”

“…The monomeric cpCV-N was shown to be moderately less stable than the wild-type CV-N and to display significantly reduced anti-HIV activity. Preliminary NMR experiments showed that cpCV-N adopts the same fold as wild-type CV-N, and the determination of a detailed, high-resolution structure is in progress [39].…”