The HIV/AIDS still remains a global health challenge with 42 million people infected
with the virus. An alarming 70% of these people reside in sub-Saharan Africa with
HIV-1 subtype C being the most prevalent subtype in this region and globally. HIV-1
protease (PR) is an obligate homodimer which plays a pivotal role in the maturation
and hence propagation of the HI virus. Although successful developments on PR
active site inhibitors have been achieved, the major limiting factor has been the
emergence of HIV drug resistant strains. It has been postulated that
disruption/dissociation of the dimer interface may lead to an inactive enzyme. The
development of small molecules and peptides has been a major research area with the
key target being the N- and C-termini antiparallel β-sheet. This is due to its highly
conserved nature and because it consists of a cluster of amino acids that contribute
most of the binding energy and stability of the dimer interface. Hence it is referred to
as a ‘hot-spot’. Therefore, binding of protease inhibitors at this site could cause
destabilisation and/or dissociation of the enzyme. The terminal residue, F99, was
mutated to an alanine disrupting the presumed lock-and-key motif it forms and in turn
creating a cavity at the N- and C-termini antiparallel β-sheet. A second mutant,
W42F/F99A, was created for monitoring tertiary structural changes exclusively at the
N- and C-termini antiparallel β-sheet. The F99A and W42F/F99A, compared to the
wild-type, showed a higher expression yield and also migrated further when separated
using tricine SDS-PAGE. Wild-type protease CD spectra showed a minimum at 214
nm and a local maximum at 230 nm, while the mutants exhibited minima at 203 nm
and absence of the local maxima. A 50% higher fluorescence intensity and a 2 nm
red-shift for the mutants versus the wild-type was observed. According to SE-HPLC
data the relative molecular weight of the wild-type, F99A and W42F/F99A are 16.4
kDa, 20.7 kDa and 18.1 kDa, respectively. Although the thermal unfolding of all three
proteases was irreversible, the unfolding transition of the wild-type was clearly
defined between 55 °C and 63 °C. The F99A and W42F/F99A unfolding curves were
linear without clearly defined transition states. The specific activity of the F99A
(0.13 μmol/min/mg) amounted to a ten-fold reduction compared to the wild-type
(1.5 μmol/min/mg). The substrate binding affinity (KM) for the F99A was 41% lower
than the wild-type when 2 μM of protein was used. The Vmax and kcat values were about 30-fold and two-fold, respectively, higher for the
wild-type when compared to the F99A. Therefore, the tricine SDS-PAGE analysis,
secondary and tertiary structural characterisation and thermal denaturation curve
showed that the F99A mutation has altered the structure causing ‘partial’ unfolding of
the protein. But, the protein still maintained minute activity. The overlap between the
ANS binding spectra of the wild-type and variants suggests that the dimeric form still
exists.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/12317 |
| Date | 29 January 2013 |
| Creators | Seele, Palesa Pamela |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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