Isothermal unfolding studies on the apo and holo forms
of Plasmodium falciparum acyl carrier protein
Role of the 4¢-phosphopantetheine group in the stability of the holo
form of Plasmodium falciparum acyl carrier protein
Rahul Modak
1
, Sharmistha Sinha
2
and Namita Surolia
1
1 Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India
2 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
Malaria continues to exact the highest mortality and
morbidity rate after tuberculosis. ‘The scourge of the
tropics’, malaria is endemic in 100 countries in the
world. Approximately 500 million cases of malaria are
reported every year, and 3000 children die of malaria
every day [1]. Our recent demonstration of the occur-
rence of the type II fatty acid synthesis (FAS) pathway
in the malaria parasite, Plasmodium falciparum, and
its inhibition by triclosan, an inhibitor of the rate-
determining enzyme of type II FAS, enoyl-acyl carrier
protein (ACP) reductase, proved the pivotal role
played by this pathway in the survival of the malarial
parasite. The essential role of fatty acids and lipids in
cell growth and differentiation, and the occurrence of
a different type (type I) of fatty acid biosynthetic
pathway in the human host from that of the malaria
parasite, make this pathway an attractive target for
developing antimalarial agents [2,3].
Keywords

acyl carrier protein-like domains, the detailed biophysical characterization
of Plasmodium acyl carrier protein can serve as a prototype for the analysis
of the conformational stability of other acyl carrier proteins.
Abbreviations
AAS, acyl-ACP synthase; ACP, acyl carrier protein; AcpS, holo-ACP synthase; apo-ACP, Plasmodium falciparum acyl carrier protein (apo
form); FAS, fatty acid synthesis; holo-ACP, Plasmodium falciparum acyl carrier protein (holo form); holo-ACP, acyl carrier protein (holo form);
LEM, linear extrapolation model; 4¢-PP, 4¢-phosphopantetheine; PfACP, Plasmodium falciparum acyl carrier protein (both apo and halo
forms).
FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS 3313
The type II FAS pathway, found in most bacteria,
plants and the malaria parasite, consists of distinct
enzymes, each catalyzing individual reactions required
to complete successive cycles of fatty acid elongation,
in contrast to the multifunctional enzyme catalyzing all
the steps of the type I FAS pathway [4,5]. ACP is an
essential component of both type I and type II fatty
acid synthesis pathways. Whereas in the type I FAS
pathway, it is an integral part of the multifunctional
enzyme, it is a discrete entity shuttling acyl groups to
the successive enzymes in the type II FAS pathway.
ACP is a small protein of molecular mass 8–10 kDa.
It plays essential roles in a myriad of metabolic path-
ways. Assorted functions involve fatty acid and lipid
biosynthesis, lipid A formation, membrane-derived
oligosaccharide biosynthesis, and activation of RTX
(repeats in toxin), toxins of Gram-negative bacteria [6–
13]. In particular instances, specialized ACPs operate
in restricted pathways such as rhizobial nodulation
signaling, and polyketide and lipoteichoic acid synthe-
sis [11,12].

pyl-b-
D-thiogalactopyranoside-induced E. coli cultures transformed with pET-28a(+)-ACP. Lane 2: protein markers; the protein bands corres-
pond to 116 kDa, 66.2 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, and 14.4 kDa (from top to bottom). Lanes 3–7: different fractions of PfACP
eluted at 50 m
M imidazole. (B) PfACP expression: native PAGE (12%) showing the ratio of holo-ACP and apo-ACP in the eluted fractions
from an Ni–nitrilotriacetic acid agarose column. Lanes 1–3: different fractions of PfACP eluted at 50 m
M imidazole. (C) Size exclusion chro-
matography profile of PfACP: holo-ACP dimer has been separated from a mixture of apo-ACP and holo-ACP monomers by size exclusion
chromatography using a Superdex 75 column (30 cm) equilibrated and eluted with 20 m
M Tris (pH 6.5) and 200 mM NaCl. Peak 1: holo-ACP
dimer. Peak 2: mixture of apo-ACP and holo-ACP monomers. (D) Separation profile of holo-ACP dimer and apo-ACP and holo-ACP mono-
mers: 12% native PAGE showing the separation of holo-ACP dimer from a mixture of apo-ACP and holo-ACP monomers. Lane 1: holo-ACP
dimer without dithiothreitol. Lane 2: holo-ACP dimer with dithiothreitol. Lane 3: mixture of apo-ACP and holo-ACP monomers. (E) Removal
of His-tag from recombinant PfACP. For the cleavage of His-tag, 1 unit of thrombin was used for 1 mg of Pf ACP at 25 °C for 2 h. On 12%
native PAGE, ACPs with and without His-tag showed significant differences in mobility. Lane 1: holo-ACP with His-tag. Lane 2: holo-ACP
without His-tag. Lane 3: mixture of holo-ACP monomer and apo-ACP with His-tag. Lane 4: mixture of holo-ACP monomer and apo-ACP with-
out His-tag. (F) Separation of apo-ACP and holo-ACP by anion exchange chromatography. Elution profile of apo-ACP and holo-ACP on a
MonoQ HR 5 ⁄ 5 anion exchange column. Peak 1: apo-ACP. Peak 2: holo-ACP. (G) Separation of apo-ACP and holo-ACP; 12% native PAGE
showing the separation of apo-ACP and holo-ACP by anion exchange chromatography. Lane 1: mixture of apo-ACP and holo-ACP. Lane 2:
purified apo-ACP. Lane 3: purified holo-ACP. (H) Dynamic light-scattering data of PfACP. (a) Particle size distribution of apo-ACP. The solid
lines indicate the accumulation percentages of particles. (b) Particle size distribution of holo-ACP. The solid lines indicate the accumulation
percentages of particles. (I) Sucrose density gradient sedimentation analysis. Forty micrograms of apo-ACP and holo-ACP were layered on
top of a 4 mL continuous 0–10% (w ⁄ v) sucrose density gradient, and this was followed by centrifugation, fractionation and 12% native
PAGE, as described in Experimental procedures. Protein bands were visualized by silver staining. (a) Lane 1: apo-ACP. Lane 2: holo-ACP.
Lanes 3–9: fractions 18–12 of sucrose density gradient for apo-ACP. Lanes 10–15: fractions 18–13 of sucrose density gradient for holo-ACP.
(b) Lanes 1, 2 and 3, respectively, are fractions 16–18 of sucrose density gradient for holo-ACP under oxidizing conditions. (c) Apo-ACP (O)
and holo-ACP (h) in each fraction was quantified by measuring the intensity of the silver-stained protein bands using
QUANTITY ONE software
and plotted against the fraction number (AU, arbitrary unit). (d) The apparent molecular masses of apo-ACP and holo-ACP were estimated on
the basis of the linear regression of the fraction number of the molecular mass markers cytochrome c (CyC), carbonic anhydrase (CA), and

sequence) was expressed in E. coli BL21 (DE3) cells
with an N-terminal His-tag. PfACP was purified by
Ni–nitrilotriacetic acid agarose affinity chromatogra-
phy to homogeneity, as shown in Fig. 1A. The purified
protein on 15% SDS ⁄ PAGE gel has a monomeric
A
E
H-a
H-b
I-c I-d
I-a I-b
FG
B
Holo-ACP dimer
123
12 3
Holo-ACP dimer
1
2
Retention time (min)
Apo-ACP with his-tag
Apo-ACP
Holo-ACP with his-tag
Holo-ACP
1234
A
280
(mAU)
1
2

1600
1400
1200
1000
800
600
400
200
0
10
Band intensity (A.U.)
12 14 16 18
34 567
1
14.4
18.4
25
35
45
66.2
116
23 45 6 7
8 9 10 11 12 13 14 15
123
23
Holo-ACP
CD
A
280
(mAU)

9417.65 Da) and 9752.831 Da (calculated 9751.65 Da)
for apo-ACP and holo-ACP, respectively [Figs 2Aa,b].
Dynamic light-scattering studies of PfACP
Apo-ACP and holo-ACP yielded hydrodynamic radii
of 1.95 ± 0.05 nm and 1.9 ± 0.1 nm, respectively,
confirming that they have a single species over the
entire experimental concentration range [Fig. 1Ha,b].
Sucrose density gradient sedimentation
In sucrose density gradient sedimentation experiments,
both apo-ACP and holo-ACP were detected between
fractions 12 and 18 [Fig. 1Ia–d]; apo-ACP showed a
major peak in fraction 15, whereas holo-ACP showed
a peak in fraction 14 [Fig. 1Ia–d]. From the calibration
curve of the sucrose density gradient, the estimated
molecular masses of apo-ACP and holo-ACP mono-
mers are $ 16.75 kDa and 21 kDa, respectively. The
dimeric peak of holo-ACP was found in major
amounts in fraction 17 when sucrose density gradient
sedimentation for holo-ACP under oxidizing condi-
tions was performed.
Fig. 2. (A). Molecular mass determination of apo-ACP and holo-ACP. Molecular masses of apo-ACP and holo-ACP were determined with an
Ultra Flex TOF ⁄ MALDI-TOF mass spectrometer. (a) Mass spectrum of holo-ACP single major peak (9752.83 Da) [holo-ACP (calculated
9751.65 Da)]. (b) Mass spectrum of apo-ACP, showing a single major peak of molecular mass 9418.84 Da [apo-ACP (calculated
9417.65 Da)]. (B) Secondary structure of ACP. The secondary structures of both apo-ACP (O) and holo-ACP (h) were determined by far-UV
CD spectroscopy. CD spectra show the presence of only a-helices as the secondary structure element in both apo-ACP and holo-ACP. (C)
Guanidine hydrochloride-induced transitions for holo-ACP (.) and apo-ACP (O)at30°C as monitored by CD at 222 nm. The proteins were in
buffer containing 5 m
M NaCl ⁄ P
i
, 100 mM NaCl and 2 mM dithiothreitol, plus the indicated concentration of guanidine hydrochloride. The solid

D
190
–1.8e+6
–2.5e+6
200
0
0
1
fraction native
[GdnHCI] M
165432
210
220 230
Wavelength (nm)
Wavelength (nm)
Molar ellipticity
240 250
–2.0e+6
–1.5e+6
–1.0e+6
–5.0e+5
0.0
5.0e+5
290
300
310
320
Wavelength (nm)
Fluorescence
330 340 350 360

0.0
2.0e+5
4.0e+5
Molar ellipticity
a
b
200 210
220
230 240 250 260
A
R. Modak et al. Plasmodium falciparum acyl carrier protein
FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS 3317
Biophysical studies with ACP
The conformations of both apo-ACP and holo-ACP at
pH 6.5 have been determined by far-UV CD spectros-
copy. Wavelength scans from 190 nm to 250 nm show
that both forms of ACP have predominantly a-helices,
which is in accordance with known ACP structures
(Fig. 2B) [24].
The conformational stability of holo-ACP and apo-
ACP was determined by chaotrope-dependent unfold-
ing at different temperatures. The reversibility of the
isothermal denaturation of ACP was shown by the
return of CD and fluorescence signals upon refold-
ing after complete denaturation with 6 m guanidine
hydrochloride (Fig. 2D,E). It was also found that the
refolded apo-ACP and holo-ACP have mobilities com-
parable to that of the nondenatured wild-type counter-
parts on 12% native PAGE, which further confirms
the reversibility of the transition (Fig. 2F). Unfolding

Æm
)1
and ) 1.97 kcalÆmol
)1
Æm
)1
,
respectively. The DG
water
values showed strong tem-
perature dependence, with maximum stability at 30 °C
(Fig. 4B).
Unfolding experiments were also monitored by the
change in fluorescence anisotropy of the single tyrosine
residue at the C-terminus of ACP. The isothermal
denaturation probed by fluorescence anisotropy corre-
lated well with the far-UV CD and fluorescence
quenching studies for both apo-ACP and holo-ACP,
further indicating that their denaturation process is
a two-state reaction (Fig. 3C). These data further
indicate that apo-ACP has lower stability than holo-
ACP.
Acyl-ACP synthesis assay with apo-ACP
E. coli holo-ACP synthase (AcpS) has been cloned and
expressed in the laboratory as a His-tagged protein.
AB
C
Fluorescence (Fit)
1.0
0.8

Far UV-CD (Fit)
Far UV-CD (Fit)
Far UV-CD (Expt)
Far UV-CD (Expt)
Fig. 3. Comparison of guanidine hydrochlo-
ride-induced transitions (A) Comparison of
guanidine hydrochloride-induced transitions
of apo-ACP at 30 °C as monitored by far-UV
CD at 222 nm (d) and tyrosine fluorescence
at 305 nm (O). (B) Comparison of guanidine
hydrochloride-induced transitions of
holo-ACP at 30 °C monitored by CD at
222 nm (m) and tyrosine fluorescence at
305 nm (n). (C) Comparison of guanidine
hydrochloride-induced transitions of
apo-ACP (O) and holo-ACP (d)at30°C
monitored by fluorescence anisotropy of
tyrosine fluorescence at 305 nm.
Plasmodium falciparum acyl carrier protein R. Modak et al.
3318 FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS
E. coli AcpS thus expressed has broad substrate specif-
icity. It utilizes apo-ACP and various acyl-CoAs as
substrates to give corresponding acyl-ACPs. This prop-
erty of AcpS was utilized to check the extent of the
reversibility of folding of apo-ACP. An acyl-ACP
synthesis assay clearly showed that both native and
refolded apo-ACP are equally and quantitatively con-
verted to lauroyl-ACP (Fig. 2G,I).
Acyl-ACP synthesis assay with holo-ACP
E. coli acyl-ACP synthase (AAS) utilizes holo-ACP as

apo-ACP
(kcalÆmol
)1
ÆM
)1
)
DG
water
holo-ACP
(kcalÆmol
)1
)
DG
water
apo-ACP
(kcalÆmol
)1
)
283 3.95 ± 0.07 3.56 ± 0.06 ) 1.55 ± 0.18 ) 1.31 ± 0.25 2.60 2.19
288 3.91 ± 0.14 3.48 ± 0.07 ) 2.12 ± 0.07 ) 1.90 ± 0.37 3.09 2.51
293 3.88 ± 0.04 3.46 ± 0.09 ) 3.12 ± 0.55 ) 1.92 ± 0.43 3.17 2.89
298 3.83 ± 0.04 3.37 ± 0.06 ) 2.37 ± 0.31 ) 1.40 ± 0.14 3.12 2.78
303 3.92 ± 0.06 3.59 ± 0.05 ) 1.67 ± 0.17 ) 1.42 ± 0.37 3.57 2.99
313 3.62 ± 0.14 3.35 ± 0.12 ) 1.95 ± 0.62 ) 1.51 ± 0.37 2.71 2.38
318 3.68 ± 0.07 3.49 ± 0.05 ) 1.75 ± 0.78 ) 1.43 ± 0.08 2.28 1.99
323 3.74 ± 0.17 ND ) 1.87 ± 0.65 ND 2.04 ND
328 3.61 ± 0.04 3.31 ± 0.09 ) 1.97 ± 0.65 ) 2.17 ± 0.37 1.86 1.08
333 3.58 ± 0.07 3.27 ± 0.05 ) 1.67 ± 0.21 ) 1.70 ± 0.37 1.01 0.74
Table 2. Average C
m

320
330
340
260
0
1
2
3
280
300
Temperature [K]
320
340
280
0
–1
–2
–3
–4
290
300
Temperature [K]
310
320
330
340
Fig. 4. Effects of temperature on the best-
fit C
m
(A), m value (B) and DG

and ACP phosphodiesterase. Although optimization of
culture conditions yields mostly holo-ACP [27], we
show that both holo-ACP and apo-ACP can be over-
expressed together and purified to homogeneity.
The secondary structures of apo-ACP and holo-
ACP, as determined by far-UV CD spectroscopy, have
shown the predominance of a-helices and a very low
percentage of b-pleated sheet in PfACP. Analysis of
CD spectra using k2d analysis software (http://www.
embl-heidelberg.de/$andrade/k2d.html) has shown
that both apo-ACP and holo-ACP contain 56%
a-helix, 10% b-pleated sheet and 34% random coil in
their secondary structure, demonstrating that PfACP
has a similar secondary structure to the other ACPs
and ACP-like domains [14–22]. Hence, detailed bio-
physical characterization of PfACP could serve as a
prototype for determining the conformational stability
of other ACPs. The NMR structure of PfACP has
been solved recently [24,25,27]; this study augments the
structural data and elucidates the interactions respon-
sible for the conformational stability of PfACP.
The size exclusion chromatography profile of PfACP
showed that the apparent molecular mass of PfACP
monomer is 25 kDa, whereas the actual molecular
masses of apo-ACP and holo-ACP are 9.4 kDa and
9.7 kDa, respectively, as is evident from MS studies.
The dynamic light-scattering experiments showed
both apo-ACP and holo-ACP exist as single species
[Fig. 1Ha,b]. The sucrose density gradient sedimenta-
tion showed that the apparent molecular masses of

unfolded by the chaotrope guanidine hydrochloride.
Detailed analyses of the stability curves obtained
by chemical denaturation are consistent with the
LEM. The chaotrope-induced equilibrium unfolding
of PfACP, followed by fluorescence, fluorescence
anisotropy and far-UV CD, showed no evidence for
the existence of stable intermediates, substantiating
the assumption of a simple two-state transition
(Fig. 3A,B). Guanidine hydrochloride-induced dena-
turation experiments on PfACP are consistent with
the LEM of protein unfolding [29].
It is apparent from the solution denaturation studies
that the holo form of the protein has greater stability
than the apo form. The differences in the unfolding
thermodynamic parameters of the two forms are given
in Tables 1–3. In the entire experimental regime, it is
seen that the holo form presents better stability than
the other form (Fig. 4C). The DG of stability is on an
average 20% greater in the case of the holoprotein as
compared to the apoprotein. Similarly, the C
m
of the
holo form always lies above the apo form at all tem-
peratures at which the experiments were conducted.
The values of T
g
, DH
g
and DC
p

g
values of the proteins vary slightly; there is a
difference of almost 6 °C between the T
g
values of the
proteins, the value being higher for the holo form.
There have been contrasting reports about the inter-
action of the 4¢-PP group with the polypeptide back-
bone and its effect on the stability of holo-ACP. The
average major conformation of the holo-ACP NMR
structure was analyzed for ligand–protein contacts
[30] ( />cgi) to determine the contacts between the 4¢-PP group
and polypeptide backbone. The greater stability of the
holo form may be due to the fact that the 4¢-PP group
(structure shown in Fig. 5A) makes a number of favo-
rable contacts with the amino acid residues at the sur-
face of the protein by virtue of the presence of several
hydrogen bond donors and acceptors in it. Further-
more, there are several hydrophobic interactions that
hold the structure firmly. Closer scrutiny of Fig. 5C
reveals that whereas most of the surface of the holo-
ACP is lined by charged residues (shown in blue ⁄ red),
the interface between the cofactor and the protein is
predominantly hydrophobic (represented by gray).
Interestingly, a few constructive interactions between
carbon and oxygen atoms were also detected at the
4¢-PP–protein interface. These favorable interactions
might result from atypical CH–O hydrogen bonds.
According to a report by Jiang et al., these atypical
hydrogen bonds play an especially crucial role in sta-

rmsd in this case happens to be 0.20 A
˚
. Again, differ-
ences are seen mostly in the loop regions where the
4¢-PP binds the protein.
In summary, our studies demonstrate that holo-ACP
has higher stability than apo-ACP. This work also
shows that the 4¢-PP group makes some contacts with
the polypeptide that stabilize the holo-ACP structure.
Experimental procedures
Chemicals and reagents
Imidazole, kanamycin, dithiothreitol, guanidine hydrochlo-
ride, thrombin from bovine plasma, sinapicnic acid, trifluor-
acetic acid, sucrose and SDS ⁄ PAGE reagents were obtained
from Sigma-Aldrich (St Louis, MO). Media components
were obtained from Difco (Franklin Lakes, NJ). All other
chemicals used were of analytical grade. All enzymes were
obtained from NEB (Ipswich, MA), MBI Fermentas GmbH
(St Leon-Rot, Germany) and Promega (Madison, WI).
Strains and plasmids
E. coli DH5a cells (Gibco BRL, Carlsbad, CA) were used
for cloning of the gene. pET-28a(+) vector (Novagen,
Darmstadt, Germany) and E. coli BL21(DE3) cells (Nov-
agen) were used for the expression of PfACP.
Cloning and expression of PfACP in E. coli
PfACP was cloned as described previously [27]. The plasmid
containing PfACP was transformed into E. coli BL21(DE3)
cells (Novagen). The culture was grown at 37 °C with vigor-
ous shaking (160 r.p.m.) in LB broth (Difco) to a cell den-
sity of D

protein was eluted using a step gradient of 50 mm to 1 m
II
II
III
IV
C
A
B
I
I
III
IV
Fig. 5. Interactions of the 4¢-PP moiety with the holo-ACP protein. The average major conformation was used for ligand–protein contact ana-
lysis. (A) The interacting atoms are labeled. The green dotted lines indicate hydrophobic interactions, and the blue lines denote CH–O hydro-
gen bonds. The 4¢-PP group is linked to the protein by the Ser37 O-c atom. Only residues 31–38 of the protein make extensive contacts
with the cofactor. For the sake of better understanding of the interactions, the entire figure has been divided into four parts (I, II, III and IV):
I, interactions with amino acids 30–33; II, interactions with amino acids 33–34; III, interactions with amino acids 35–37; IV, interactions with
amino acids 37–38. (B) The overlay of the apo (green) and holo (orange) forms of the protein (rmsd ¼ 0.20 A
˚
). (C) Diagram showing the nat-
ure of the surface in holo-ACP. It should be noted that the protein has a greater number of charged exposed surface (indicated by blue ⁄ red)
than hydrophobic ones. The red line denotes the area in the protein that makes contact with the cofactor.
Plasmodium falciparum acyl carrier protein R. Modak et al.
3322 FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS
imidazole, and fractions were tested for purity by 15%
SDS ⁄ PAGE. The ratio of apo-ACP and holo-ACP was
checked by 12% native PAGE. Purified PfACP (2 mgÆmL
)1
)
was injected onto a Superdex 75 HR 10 · 300 mm column

(pH 6.5) and 2 mm dithiothreitol, and eluted with an NaCl
gradient in the same buffer [33]. Finally, both apo-ACP
and holo-ACP were subjected to buffer exchange in 5 mm
Na ⁄ K phosphate (pH 6.5), 100 mm NaCl and 2 mm dithio-
threitol using a HI-Trap desalting column, and stored at
) 80 °C until further use.
PAGE under native conditions
Samples were mixed with 6· sample loading buffer (300 mm
Tris, pH 6.8, 0.6% bromophenol blue, 60% glycerol) and
were analyzed by 12% PAGE without SDS. The electro-
phoresis was performed at room temperature under a con-
stant current of 25 mA per gel. The gels were stained with
Coomassie Blue. Urea PAGE for conformation-sensitive
PAGE was prepared similarly, except for the addition of
5 m urea and an increase in the acrylamide concentration to
20% [34]. The sample buffer also contained 2.5 m urea.
Determination of molecular masses of apo-ACP
and holo-ACP
Purified holo-ACP and apo-ACP were desalted in water
using a Hi-Trap desalting column (Amersham Biosciences).
Samples were mixed uniformly with 1 lL of the matrix, pre-
pared by adding 0.05% trifluoroacetic acid to a saturated
solution of sinapinic acid (3,5-dimethoxy-4-hydroxycinnam-
ic acid), and spotted onto the MALDI plate. The molecular
mass was determined with an ULTRA FLEX TOF ⁄
MALDI-TOF mass spectrometer from Bruker Daltonics
(Bremen, Germany).
Biophysical characterization of ACP
CD spectroscopy
CD spectra in the far-UV region (200–250 nm) were collec-

25 °C in a 1 mL quartz cuvette, by exciting the samples
at 280 nm and recording the emission between 295 and
350 nm.
Fluorescence anisotropy experiments
The anisotropy experiments for the single tyrosine residue
at the C-terminus were performed to study equilibrium
guanidine hydrochloride-dependent denaturation. A Jobin-
Yvon spectrofluorimeter with an excitation slit width of
2 nm and emission of 5 nm was used. Samples were excited
at 280 nm, and emission was recorded at 305 nm. Apo-
ACP and holo-ACP at 60 lm were used for the studies.
The anisotropy was calculated according to the following
equation [36]:
A ¼
I
q
À I
?
I
P
þ 2I
?
where I
||
is the fluorescence in the parallel direction, and I
^
is the fluorescence in the perpendicular direction.
R. Modak et al. Plasmodium falciparum acyl carrier protein
FEBS Journal 274 (2007) 3313–3326 ª 2007 The Authors Journal compilation ª 2007 FEBS 3323
Data analysis

In the first method, a single guanidine hydrochloride-
induced denaturation, where the observed CD signal at
each point in the unfolding experiment is m
obs
, was
analyzed with the equation
where N
o
and D
o
represent the intercept and a
N
and a
D
represent the slopes of the folded and unfolded baselines,
respectively. This expression combines the LEM (Eqn 5),
[29] where DG
H2O
¼ ) mC
m
, the two-state assumption for
the unfolding reaction, and linear pretransition and post-
transition baselines, which are dependent on the concentra-
tion of guanidine hydrochloride by the equation X
o
+ a
X
[guanidine hydrochloride].
In the second method of analysis, the raw data were first
converted to plots of f

1 À fu
ð4Þ
DG
0
¼ÀRTlnKeq ð5Þ
The unfolding of a protein is accompanied by the exposure
of the hydrophobic core region, which is reflected in
the change in heat capacity, C
p
. In order to calculate the
change in heat capacity (DC
p
) for the reaction, we used the
method of Pace, where the free energies (DG
o
) calculated at
different temperatures are fitted to the Gibbs–Helmholtz
equation.
DGðTÞ¼DH
g
ð1 À T=T
g
ÞþDC
p
½T À T
g
À T lnðT=T
g
Þ ð6Þ
Acyl-ACP synthesis assay for apo-ACP

and 200 lm lauric acid were used per assay. The reaction
mixtures were incubated at 37 °C for 2 h, and the product
was checked by 20% conformation-sensitive PAGE with
5 m urea [36].
Dynamic light-scattering studies of PfACP
Dynamic light-scattering studies were performed on a
Brookhaven Instruments (Holtsville, NY, USA) Dynamic
Light Scattering set-up that can measure sizes from 2 to
m
o
obs
¼
N
o
þ a
N
½GdnHClþðD
0
þ a
D
½GdnHClÞ Â exp ½
m
RT
ð½GdnHClÀCmÞ
1 þ exp½
m
RT
ð½GdnHClÀCmÞ
ð2Þ
Plasmodium falciparum acyl carrier protein R. Modak et al.

quantified by quantity one (BioRad, Hercules, CA, USA)
software.
Acknowledgements
This work was supported by the Department of Bio-
technology and the Department of Science and Tech-
nology, Government of India to N. Surolia. The
authors wish to thank Dr Siddharth Sarma and Alok
Sharma for discussions.
References
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2 Surolia N & Surolia A (2001) Triclosan offers protec-
tion against blood stages of malaria by inhibiting enoyl-
ACP reductase of Plasmodium falciparum. Nat Med 7,
167–173.
3 Kapoor M, Dar MJ, Surolia A & Surolia N (2001)
Kinetic determinants of the interaction of enoyl-ACP
reductase from Plasmodium falciparum with its
substrates and inhibitors. Biochem Biophys Res Commun
289, 832–837.
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