FOLDING OF THE MULTIDOMAIN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-I INTEGRASE

Protein folding conditions were established for human immunodeficiency virus integrase (IN) obtained from purified bacterial inclusion bodie s, IN was denatured by 6 M guanidine.HCl-5 mM dithiothreitol, purified by gel filtration, and precipitated by ammonium sulfate. The reversib le solvation of precipitated IN by 6 M guanidine.HCl allowed for wide variation of protein concentration in the folding reaction. A 6-fold d ilution of denatured IN by 1 M NaCl buffer followed by dialysis produc ed enzymatically active IN capable of 3' OH end processing, strand tra nsfer, and disintegration using various human immunodeficiency virus-1 (HIV-1) long terminal repeat DNA substrates. The specific activities of folded IN preparations for these enzymatic reactions were comparabl e to those of soluble IN purified directly from bacteria. The subunit composition and enzymatic activities of IN were affected by the foldin g conditions. Standard folding conditions were defined in which monome rs and protein aggregates sedimenting as dimers and tetramers were pro duced. These protein aggregates were enzymatically active, whereas mon omers had reduced strand transfer activity. Temperature modifications of the folding conditions permitted formation of mainly monomers. Upon assaying, these monomers were efficient for strand transfer and disin tegration, but the oligomeric state of IN under the conditions of the assay is indeterminate. Our results suggest that monomers of the multi domain HIV-1 IN are folded correctly for various catalytic activities, but the conditions for specific oligomerization in the absence of cat alytic activity are undefined.