       Document 0041
 DOCN  M95A0041
 TI    Catalytic domain of human immunodeficiency virus type 1 integrase:
       identification of a soluble mutant by systematic replacement of
       hydrophobic residues.
 DT    9510
 AU    Jenkins TM; Hickman AB; Dyda F; Ghirlando R; Davies DR; Craigie R;
       Laboratory of Molecular Biology, National Institute of Diabetes; and
       Digestive and Kidney Diseases, Bethesda, MD 20892-0560, USA.
 SO    Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6057-61. Unique Identifier
       : AIDSLINE MED/95320214
 AB    The integrase protein of human immunodeficiency virus type 1 is
       necessary for the stable integration of the viral genome into host DNA.
       Integrase catalyzes the 3' processing of the linear viral DNA and the
       subsequent DNA strand transfer reaction that inserts the viral DNA ends
       into host DNA. Although full-length integrase is required for 3'
       processing and DNA strand transfer activities in vitro, the central core
       domain of integrase is sufficient to catalyze an apparent reversal of
       the DNA strand transfer reaction, termed disintegration. This catalytic
       core domain, as well as the full-length integrase, has been refractory
       to structural studies by x-ray crystallography or NMR because of its low
       solubility and propensity to aggregate. In an attempt to improve protein
       solubility, we used site-directed mutagenesis to replace hydrophobic
       residues within the core domain with either alanine or lysine. The
       single substitution of lysine for phenylalanine at position 185 resulted
       in a core domain that was highly soluble, monodisperse in solution, and
       retained catalytic activity. This amino acid change has enabled the
       catalytic domain of integrase to be crystallized and the structure has
       been solved to 2.5-A resolution [Dyda, F., Hickman, A. B., Jenkins, T.
       M., Engelman, A., Craigie, R. & Davies, D. R. (1994) Science 266,
       1981-1986]. Systematic replacement of hydrophobic residues may be a
       useful strategy to improve the solubility of other proteins to
       facilitate structural and biochemical studies.
 DE    Amino Acid Sequence  Binding Sites  Catalysis  Chromatography, Gel
       Comparative Study  DNA Nucleotidyltransferases/*CHEMISTRY/ISOLATION &
       PURIF/  *METABOLISM  Electrophoresis, Polyacrylamide Gel  Enzyme
       Stability  Histidine  HIV-1/*ENZYMOLOGY/GENETICS  Macromolecular Systems
       Models, Molecular  Molecular Sequence Data  *Mutagenesis, Site-Directed
       *Point Mutation  *Protein Conformation  Recombinant
       Proteins/CHEMISTRY/ISOLATION & PURIF/METABOLISM  Sequence Tagged Sites
       Solubility  Support, U.S. Gov't, P.H.S.  Virus Integration  JOURNAL
       ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

