The
nucleobase/ascorbate
transporters
(NAT)
are
responsible
for
the
uptake
of
nucleobases
in
all
kingdoms
of
life,
and
for
transportation
of
ascorbic
acid
(vitamin
C)
in
humans.
Nucleobase
analogues
are
often
used
as
antimetabolites
in
the
treatment
of
a
range
of
diseases.
A
detailed
understanding
of
nucleobase
transport
will
increase
understanding
of
the
mechanism
of
action
of
these
important
proteins
and
facilitate
design
of
drugs
with
enhanced
efficacy
and
specificity.
Owing
to
the
extensive
genetic
and
biochemical
analysis,
UapA
from
A.
nidulans
is
the
best
studied
example
of
the
NAT
family.
The
aim
of
this
project
was
to
produce
pure
and
stable
UapA
from
A.
nidulans
for
structural
studies
by
X-­‐ray
crystallography.
In
this
study,
it
was
possible
to
express
wild
type
UapA
in
S.
cerevisiae
as
a
GFP
fusion
to
1.91
mg/L.
Following
purification,
the
protein
was
extremely
unstable,
degrading
almost
completely
after
48
hours
at
4°C.
A
number
of
single
point
mutants
previously
shown
to
exhibit
reduced
transport
activity
but
correct
targeting
to
the
membrane
were
generated
in
an
attempt
to
increase
UapA
stability.
hFSEC
and
FSEC
allowed
successful
identification
of
the
thermostable
mutant
G411V,
with
preserved
xanthine
binding.
G411V
expressed
well
as
a
GFP
fusion
and
was
purified
to
homogeneity
with
a
yield
of
2.8
mg
from
10
L
culture.
The
purified
protein
was
stable
at
4°C
for
at
least
2
days.
Removal
of
the
11
amino
acid
residues
at
the
N
terminus,
G411V12-­‐574,
improved
purification
yield
to
4.2
mg
from
10
L
culture
and
resulted
in
protein
that
was
stable
at
4°C
for
at
least
6
days.
Optimised
crystals
of
this
construct
currently
diffract
to
7.16
Å
resolution.
Boron
(B)
toxicity
and
deficiency
in
plants
are
worldwide
agricultural
problems.
Understanding
the
mechanism
of
boron
homeostasis
in
plant
will
provide
route
for
the
design
of
crops
tolerant
to
soils
with
suboptimal
B
concentrations.
We
aimed
to
solve
the
structure
of
a
boric
acid
transporter
from
A.
thaliana.
Preliminary
crystals
diffracted
to
7.6
Å.
Significant
optimisation
screening
including
the
use
of
structure
specific
antibodies
failed
to
improve
the
diffraction
limit.
A
breakthrough
came
with
the
use
of
a
novel
type
of
amphiphile,
undecyl-­‐MNG,
which
significantly
improved
the
diffraction
limit
to
5.0
Å.
Crystal
optimisation
is
underway
to
further
improve
the
resolution.