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well folded since it is relatively resistant to proteolysis. As it is also active for disintegration, we
concluded that it contained the enzyme active site.
Page 93
We exploited the convenience of histidine-tag (HT) technology to develop methods to purify large
quantities of IN 50–212 [12]. A 20-amino-acid histidine-containing tag was added to the N-terminus of
HIV-1 IN 50–212 [22] to allow rapid purification on nickel affinity columns. It was subsequently removed
by thrombin cleavage. Biophysical studies showed that although buffer conditions could be identified
where the protein was soluble to ~ 4 mg/mL, under these conditions the protein was highly aggregated
(unpublished observations). Although the aggregation problem could be largely avoided by the addition
of high concentrations of the zwitterionic detergent CHAPS, conditions could not be identified under
which protein crystals formed in the presence of CHAPS.
D. Systematic Mutation of Hydrophobic Residues to Improve Protein Solubility
As it became clear that IN 50–212 was crystallographically challenged, a condition readily understood in
terms of its aggregation problems and low solubility, a more radical approach was undertaken to try and
improve its biophysical properties. Hydrophobic residues in the catalytic core were targeted for sitespecific
mutation according to the following criteria: where two or more hydrophobic residues were
encountered close together in the primary amino acid sequence, they were each changed to an alanine
residue. When a hydrophobic residue stood alone, it was mutated to lysine. In this way, 29 different
mutant proteins of IN 50–212 were rapidly generated using the overlapping polymerase chain reaction
(PCR) and screened for improved solubility properties [34]. Three mutated proteins were identified that
were more soluble at lower NaCl concentration than the unmutated core (V165K, F185K, and the
double mutation of W131A/W132A). However, one of these in which Phe185 was mutated to Lys had
dramatically improved solubility and was ultimately crystallized and its three-dimensional structure
determined [3]. The remarkable biophysical properties of this single point mutant of IN 50–212 have
recently been described [34].
IV. Structure Of The Catalytic Core Domain Of HIV-1 Integrase
A. Description of the Structure
The Overall Protein Fold
The three-dimensional structure of the catalytic core domain of HIV-1 integrase is centered on a mixed
five stranded β sheet flanked by several helices forming
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