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Design and initial characterization of a circular permuted variant of the potent HIV‐inactivating protein cyanovirin‐N
Author(s) -
Barrientos Laura G.,
Louis John M.,
Hung Jason,
Smith Trent H.,
O'Keefe Barry R.,
Gardella Roberta S.,
Mori Toshiyuki,
Boyd Michael R.,
Gronenborn Angela M.
Publication year - 2001
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.10024
Subject(s) - mutant , monomer , wild type , circular dichroism , protein secondary structure , protein folding , denaturation (fissile materials) , mutant protein , chemistry , molecule , crystallography , biology , biochemistry , gene , organic chemistry , nuclear chemistry , polymer
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. Proteins 2002;46:153–160. © 2001 Wiley‐Liss, Inc.