Báo cáo khoa học: Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu docx

Chaperone activity of recombinant maize chloroplast protein synthesis
elongation factor, EF-Tu
Damodara Rao
1
, Ivana Momcilovic
1
, Satoru Kobayashi
1
, Eduardo Callegari
2
and Zoran Ristic
1
1
Department of Biology, University of South Dakota and
2
Department of Basic Biomedical Sciences, University of South Dakota
School of Medicine, Vermillion, SD, USA
The p rotein synthesis e longation factor, EF-Tu, is a protein
that carries aminoacyl-tRNA to the A -site o f the ribosome
during t he elongation phase of protein synthesis. In maize
(Zea mays L) th is protein has been implicated in heat tol-
erance, andit has been hypothesized that EF-Tu confers heat
tolerance by a cting as a molec ular chaperone and protecting
heat-labile proteins from thermal aggregation and inactiva-
tion. In this study w e i nvestigated t he effect of the recom-
binant precursor of maize EF-Tu (pre-EF-Tu) o n t hermal
aggregation and inactivation of t he heat-labile proteins, c it-
rate synthase and malate dehydrogenase. T he recombinant
pre-EF-Tu was purified from Escherichia coli expressing
this protein, and m ass s pectrometry confirmed that the
isolated protein was indeed maiz e EF-Tu. The purified

protein was capable of binding GDP (indicative of protein
activity) and was stable at 45 °C, the highest temperature
used in this study to test this protein f or possible c haperone
activity. Importantly, t he recombinant m aize pre-EF-Tu
displayed chaperone activity. It protected citrate synthase
and malate dehydrogenase from th ermal aggregation and
inactivation. To our knowledge, this is the first observation
of chaperone activity by a plant/eukaryotic pre-EF-Tu
protein. The results of this study support the hypothesis that
maize EF-Tu plays a role in heat tolerance b y a cting a s a
molecular chaperone and protecting chloroplast proteins
from thermal aggregation a nd inactiv ation.
Keywords: chloroplast protein synthesis elongation factor
(EF-Tu); chaperones ; heat stress; heat tolerance; Zea mays.
Chloroplast protein s ynthesis elongation factor, EF-Tu, i s a
protein (45–46 kDa) that plays a key role i n t he elongation
phase of protein synthesis [1–3]. This p rotein catalyzes the
GTP-dependent binding of aminoacyl-tRNA to the A-site
of the ribosome [ 3]. In land plants, EF-Tu is encoded b y the
nuclear genome and synthesized in the cytosol [4]. Chloro-
plast EF-Tu is highly conserved, and it shows a high
sequence similarity to prokaryotic EF-Tu [3,5].
Studies from our laboratory have s uggested that in maize
(Zea mays L) chloroplast EF-Tu may play a role in the
development of heat tolerance. The evidence for this
conclusion includes: (a) positive correlation between the
heat-induced accumulation of EF-Tu and plant ability to
tolerate heat stress in several genotypes of maize [5–7], (b)
association between the heat-induced synthesis of EF-Tu
and the maize heat t olerance phenotype [8], (c) increased

tolerance to heat st ress in Escherichia coli expressing maize
EF-Tu [9], (d) decreased t olerance to heat stress in a m aize
mutant with reduced capacity to accu mulate EF-Tu [10],
and (e) increased thermal stability o f chloroplast p roteins in
maize with higher levels o f EF-Tu [10,11]. (It should be
noted that in the previous studies [6–8] maize EF-Tu was
referred to as a 45–46 kDa heat shock p rotein because the
identity of this pro tein was not known until the r eport of
Bhadula et al . [5].)
A h ypothesis h as been developed that m aize EF-Tu may
confer heat to lerance by protecting o ther proteins from
heat-induced aggregation and inactivation (thermal dam-
age), thus acting as a molecular chaperone [10,11]. I n t his
study we investigated the effect of th e recombinant p recur-
sor of m aize EF-Tu (pre-EF-Tu, w hich has a 58 amino acid
long chloroplast targeting sequence a t the N-terminal end
[5]) on thermal aggregation and inactivation of the heat-
labile proteins, citrate synthase (CS) and malate dehydro-
genase (MDH). H ere we r eport, for the firs t time, that t he
recombinant maize pre-EF-Tu displays ch aperone proper-
ties, as it protected heat-labile proteins from thermal
damage.
Materials and methods
Expression of maize pre-EF-Tu in
Escherichia coli
E. coli expressing maize pre-EF-Tu was previously trans-
formed [9] using a cDNA for maize (Z. mays L) EF-Tu,
designated as Zme ftu1 [5]. Zme ftu1 was s ubcloned into the
expression vector pTrcHis2A, which adds C-terminal c-myc
and polyhistidine tags to the protein, and the pTrcHis2A

vector carrying Zmeftu1 w as used t o transform competent
E. coli cells of the s train DH5 a [9].
In the c urrent study, the induction of expression of maize
pre-EF-Tu in E. coli was carried o ut according to Moriarty
et al . [9]. F ollowing induction, the r ecombinant protein was
isolated and purified from the E. coli culture.
Correspondence to Z. Ristic, Department of Biology, University of
South Dakota, Vermillion, SD 57069, U S A. Fax: +1 605 677 6557,
E-mail:
Abbreviations: CS, citrate synthase; MDH, malate dehydrogenase;
HSPs, heat shock proteins; sHSPs, small heat shock proteins.
(Received 5 May 2004, revised 1 4 July 2 004, accepted 27 July 200 4)
Eur. J. Biochem. 271, 3684–3692 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04309.x
Isolation and purification of recombinant pre-EF-Tu
from
E. coli
E. coli cells expressing maize p re-EF-Tu were collected by
centrifugation (50 mL cell c ulture; 10 000 g,30min),
washed, and suspended in isolation buffer [20 m
M
Tris/
HCl pH 8.0 containing 20 m
M
NH
4
Cl, 10 m
M
MgCl
2
,

2m
M
dithiothreitol, 0.1 m
M
EDTA, 10% (v/v) glycerol,
1m
M
phenylmethanesulfonyl fluoride] according to S tanzel
et al . [12]. Crude protein extract from harvested cells was
then p repared by the lysozyme/EDTA method [13]. Cells
were sonicated at a medium intensity setting, holding the
suspension on ice. After sonication, i nsoluble debris w as
removed by centrifugation at 5000 g for 15 min. The
supernatant (lysate) w as then passed through a 0.8 lm
syringe filter and stored at )70 °C until further use.
Purification of recombinant pre-EF-Tu was c arried out
according to Stanzel et al. [12]. Recombinant protein was
purified by SP-Sepharose, Q-Sepharose and gel filtration
chromatography. SP-Sepharose was packed in a column
(25 cm · 1 cm), equilibrated w ith e ight column volumes o f
20 m
M
acetate buffer (pH 4.8) consi sting of 10 m
M
MgCl
2
,
2m
M
dithiothreitol, 0.1 m

M
EDTA, 10% (v/v) glycerol,
and 1 m
M
phenylmethanesulfonyl fluoride. The fractions
from the S P-Sepharose column were analyzed by 1D SDS/
PAGE, and fractions having prominent bands between
45 kDa and 55 kDa were pooled [typically, pooled fractions
had a total of four to five bands with molecular masses
ranging f rom 20 t o 100 kDa but the prominent bands (1–2)
were between 45 and 55 kDa range] and d ialyzed against
isolation buffer overnight. A fter dialysis , t he dialysate was
applied to Q-Sepharose column (30 cm · 1 cm), f ollowed
by washing the column with the same buffer. The bound
recombinant pre-EF-Tu was eluted with a linear gradient of
0.0–0.5
M
NaCl in the isolation buffer. Fractions (2 mL)
were collected and an alyzed by 1D SDS/PAGE. The
fractions containing purified pre-EF-Tu were pooled and
concentrated using a centrifuge filter device Amicon )50
(Millipore, Bedford, MA). The concentrated protein was
then applied to Sephacryl SS-100 (50 cm · 2.5 cm), eluted
with the isolation buffer, and protein concentration was
determined using the Br adford Ass ay (Bio-Rad, CA). The
purity of r ecombinant pre-EF-Tu prepar ation was checked
using 1 D SDS/PAGE and Western blotting [5], the identity
of the purified protein was verified using mass spectrometry,
and the ability of the protein to bind GDP (indicative of
EF-Tu activity [14]) was assessed using the [

3
H]GDP
exchange assay [14,15]. In addition, the heat stability of
purified pre-EF-Tu was also assessed as described below.
One-dimensional SDS/PAGE and Western blotting
One-dimensional SDS/PAGE of purified recombinant pre-
EF-Tu was carried out according to Laemmli [16]. In
separate trials, 1D SDS/PAGE g els were s tained using
Coomassie Brilliant Blue R250 and Silver stain (Amersham).
Western blot analysis w as performed as described b y
Moriarty et al. [9]. The purified protein was resolved on
10% (w/v) polyacrylamide gel with SDS, and then trans-
ferred to a nitrocellulose membrane (Bio-Rad, Hercules,
CA). The blot w as probed for recombinant p rotein using
the E CL che miluminescent development me thod with
primary antibody raised against the my c epitope, which is
included near the C-terminus of recombinant protein [9].
Mass spectrometry
Mass spectrometry analysis was performed according to Koc
et al . [17]. Purified protein was separated on a 10% (w/v)
polyacrylamide gel with SDS and stained by Coomassie
Brilliant Blue R250. The stained protein band was then
excised from t he gel, and the protein spot was digested in-gel
with Tryp sin (Promega, Madison, WI) [18]. The peptides
produced were injected in to a C apillary LC (Waters
Corporation, Milford, MA) to be desalted and separated
using a C18 RP PepMap, 75 lm (internal diameter) column
(LC P ackings, Dionex, San Francisco, CA). The standard
gradient us ed was a s follows: 0 –2 min, 3% B isocratic;
2–40 min, 3–80% B linear. Mobile phase A w as water/

acetonitrile/formic acid ( 98.9 : 1 : 0.1, v /v/v), and phase B
was acetonitrile/water/formic acid (99 : 0.9 : 0.1, v/v/v). The
total solvent fl ow w as 8 lLÆmin
)1
. Samples were analyzed
under nano-ESI/MS and nano-ESI-MS/MS u sing a Q-TOF
micro mass spectrometer (Micromass, Manchester, UK).
The s pectrum data were acquired by
MASSLYNX
4.0 s oftware
(Micromass), and peptide matching and protein searches
were performed auto matically using the
PROTEINLYNX
1.1
Global Server ( Micromass). T he peptide m asses a nd
sequence tags were searched against the NCBI nonredundant
protein database.
[
3
H]GDP exchange assay
The a ctivity of recombinant pre-EF-Tu was assessed b y the
[
3
H]GDP exchange assay [14]. Various amounts of purified
protein were added to scintillation vials containing 40 lLof
binding buffer ( 250 m
M
Tris/HCl, pH 7.4, 50 m
M
MgCl

2
,
250 m
M
NH
4
Cl, 25 m
M
dithiothreitol) and 4.5 nCi of
[
3
H]GDP (specific activity 27.7 mCi Æmg
)1
or 12.3 CiÆ
mmol
)1
; t otal volume of reaction mixture, 200 lL). As
controls, BSA an d ovalbumin (Sigma) were used. The
reaction mixtures were allowed to equilibrate for 10 m in at
37 °C then were diluted with 2 mL of wash buffer (10 m
M
Tris/HCl pH 7.4, 10 m
M
MgCl
2
,10m
M
NH
4
Cl), filtered

using Millipore discs (pore s ize, 0.2 lm; diameter, 5 0 mm),
and washed t hree times w ith 3 mL of the same buffer . The
filters were dissolved in 5 mL of scintillation fluid, and the
radioactivity w as monit ored u sing a Beckman LS 6500
scintillation counter.
Heat stability of recombinant pre-EF-Tu
The h eat stability of r ecombinant pre-EF-Tu was assessed
using two approaches. In the first approach, 1 mL samples
of purified protein (0.5 l
M
) were incubated at 25 °C
(control) or 45 °C (heated) for 45 min. After incubation,
samples were centrifuged and the supernatant of each
sample (control a nd heated) was analyzed for t he presence
of pre-EF-Tu using Western blotting and anti-myc Ig as
described above. Equal volumes of protein s amples were
loaded in each gel lane. In the second approach, 1 mL
aliquot of recombinant protein (0.5 l
M
) was incubated at
45 °C for 45 min i n c overed quartz cuvettes, and t he heat
stability o f EF-Tu was a ssessed by monitoring light
Ó FEBS 2004 Chaperone activity of recombinant maize EF-Tu (Eur. J. Biochem. 271) 3685
scattering at 320 nm during incubation [11,19]. As a control,
a heat-labile p rotein, citrate synthase, [20] was used. In
addition, the a ctivity of recombinant pre-EF-Tu was also
measured af ter heating (45 min at 45 °C) by the [
3
H]GDP
exchange assay [14] as outlined above.

Chaperone assays
The recombinant pre-EF-Tu was t ested for possible chap-
erone activity using t wo approaches: (a) by monitoring
thermal aggregation of heat-labile CS or MDH in the
presence or absence of pre-EF-Tu, and (b) by measuring
residual activity o f CS or MDH after heating in the presence
or absence o f pre-EF-Tu. CS and MDH w ere chosen as
model s ubstrates because they are known to be relatively
heat-labile and have b een used in chaperone studies [20–23].
CS and MDH were obtained from Boehringer Mannheim.
Both enzymes are homodimers, and in the t ext and figures
the c oncentrations of CS and MDH refer to the 98 kDa
homodimer and 70 kDa homodimer, respecti vely.
The thermal aggregation of CS and MDH was tested
according to Lee et al . [ 21]. In separate trials, CS (0.15 l
M
)
and MDH (0.3 l
M
) were m ixed with various amounts of
purified recombinant pre-EF-Tu (as indicated in Fig. 6 ) in
20 m
M
Tris/HCl buffer [7 m
M
MgCl
2
,60m
M
NH

4
Cl,
0.2 m
M
EDTA, and 10% (v/v) glycerol; pH 8; total v olume
1 m L] in covered quartz cuvettes. Three controls were used:
CS o r MDH a lone, CS or M DH mixed with BSA, a nd CS
or MDH mixed with ovalbumin (molar concentrations are
indicated in F ig. 6 ). Samples were incubated at indicated
high temperatur es (CS: 41 °Cor45°C; MDH: 45 °C) for
45 min, and CS or MDH stability was estimated by
monitoring light scattering at 320 nm during incubation
[21].
The residual activity of CS a nd MDH w as determined
using the experimental d esign of L ee et al. [21]. In separate
trials, CS ( 2 l
M
)andMDH(0.5l
M
)weremixedwith4l
M
and 2 l
M
of purified recombinant pre-EF-Tu, respectively.
Aliquots (1 mL) of the mixtures [CS mixture: 0.2 m
M
acetyl-CoA, 0.5 m
M
oxaloacetic acid, 0.1 m
M

5,5¢-dithio-
bis(2-nitrobenzoic acid) in 100 m
M
Tris/HCl (pH 7 .5);
MDH mixture : 0 .1 m
M
NADH, 0.1 m
M
oxaloacetic acid
in 50 m
M
Tris/HCl (pH 7.5)] were then heated at various
high temperatures ( 38 °C, 41 °C, 43 °C, and 45 °C) for
30 min. As controls, C S or M DH alone and CS or MDH
mixed with B SA or ovalbumin w ere used (molar concen-
trations for BSA and ovalbumin are indicated in Fig. 7).
After heating, aliquots were quickly cooled to room
temperature and then kept on ice for up to 75 min
(75 m in recovery). The residual activity of CS a nd MDH
was measured at room temperature immediately after
heating and at various times of r ecovery, according to S rere
[24] and Banaszak & Bradshaw [25], respectively.
We also investigated the possible e ffect of recombinant
pre-EF-Tu on reactivation of heat-inactivated CS and
MDH. In separate trials, C S (2 l
M
)andMDH(0.5l
M
)
were incubated at 43 °C for 30 min, without the presence of

pre-EF-Tu. I mmediately after incubation, pre-EF-Tu was
added to the heated pr otein s amples (molar concentrations
for pre-EF-Tu are indicated in F ig. 7) and the r eaction
mixtures were allowed t o recover for 4 5 min. The mixtures
were kept on ice during recovery. The residual activity of CS
and MDH was t hen m easured at various times of recovery
as described above.
Results
One-dimensional SDS/PAGE, Western blot, and mass
spectrometry analysis of purified recombinant pre-EF-Tu
The r ecombinant m aize pre-EF-Tu was purified to homo-
geneity f rom E. coli expressing this protein [ 9]. A previou s
study has shown that the expressed maize pre-EF-Tu
appears in E. coli in a highly s oluble form [ 9]. Both s ilver
stained ( Fig. 1, lane 1) and Coomassie B lue s tained (Fig . 1,
lane 2) 1D SDS/PAGE gels showed a single band indicating
purified protein. The molecular mass of the purified protein
was approximately 50–51 kDa; this molecular mass was
greater than that of the native chloroplast EF-Tu
(45–46 k Da) [5] because of the presence of a chloroplast
targeting sequence a t the N-terminal end a nd the c-myc and
polyhistidine tags at the C-terminus o f the polypeptid e [9].
Western b lot probed with anti-myc Ig, which is specific to
recombinant pre-EF-Tu carrying the c- myc tag [9], also
showed a single band with the molecular mass of
50–51 kDa (Fig. 1, lane 3). The identity of the purified
protein was further c onfirmed by m ass spectrometry, which
showed that the purified protein was indeed chloroplast
pre-EF-Tu (Fig. 2). The recombinant p rotein amino acid
sequence obtained by mass spectrometry (Fig. 2B,C)

matched t he sequence of maize chloroplast EF-Tu [5] and
chloroplast EF-Tu from Oryza s ativa L., Glycine m ax (L)
Merr, Pisum sativum L., and Nicotiana silvestris Speg.
(NCBI nonredundant protein database; data not shown).
GDP binding activity of purified recombinant pre-EF-Tu
We assessed the activity of purified pre-EF-Tu using the
[
3
H]GDP exchange assay [14]. The assay showed that the
Fig. 1. One-dimensional SDS/PAGE gels and Western blot o f purified
recombinant maizepre-EF-Tu.The recombinant protein was purified
from E. coli expressing this protein. Lane 1, gel stained with silver
stain; lane 2, gel stained with Coomassie Brilliant Blue R250; lane 3 ,
Western blot probed w ith a nti-myc Ig. A rrow indicates r ecombinant
pre-EF-Tu ( 50–51 kDa). Note: E. c oli EF-Tu has a mass of 43 kDa
[22], and the protein band of this mass is not seen in lanes 1 and 2.
Hence, th e purified protein s een in lanes 1 and 2 is most p robably
maize pre-EF-Tu.
3686 D. Rao et al.(Eur. J. Biochem. 271) Ó FEBS 2004
purified pre-EF-Tu binds [
3
H]GDP (Fig. 3) suggesting t hat
this protein was probably i n a phys iologically active form.
As indicated by an increase i n radioactivity (disintegration
per m inute), the binding of pre-EF-Tu with GDP increased
with an increase in the concentration o f recombinant
protein (Fig. 3). No significant radioactivity, however, w as
detected when [
3
H]GDP was mixed with control proteins,

BSA or ovalbumin (Fig. 3 ).
Heat stability of recombinant maize pre-EF-Tu
The h eat stability of r ecombinant pre-EF-Tu was assessed
as its ability t o remain soluble and maintain activity at high
temperature [20]. L ight scattering experim ents with heated
aliquots of purified pre-EF-Tu showed that this protein w as
heat stable (remained soluble) at 45 °C, as no increase in
relative light scattering was observed when the protein was
heated at this temperature ( Fig. 4A) . The control protein
(CS), in c ontrast, showed no stability at 4 5 °C, indicated by
an increase in relative light scattering (Fig. 4 A). The pre-
EF-Tu also maintained its activity a t h igh t emperature
(45 °C). As indicated by the [
3
H]GDP exchange assay, the
binding of h eated pre-EF-Tu with GDP was s imilar to
the binding of nonheated (25 °C) pre-EF-Tu with GDP
(Fig. 3 ). Western blot analysis of the supernatant of
centrifuged heated samples of purified pre-EF-Tu corro-
borated the results of light the scattering experiments. A
Western b lot revealed t hat t he recombinant pre-EF-Tu was
present in the soluble fraction (supernatant) at 45 °C,
indicating its stability at this temperature (Fig. 4B).
Recombinant maize pre-EF-Tu protected CS and MDH
from thermal aggregation
The recombinant maize pre-EF-Tu protected CS from
thermal aggregation. When heated at either 41 °Cor45°C,
CS began to form insoluble aggregates, indicated by an
increase in relative light scattering (Fig. 5A,B). T he CS
aggregation, however, was reduced in the presence o f

various amounts of pre-EF-Tu and was almost completely
suppressed a t a n pre-EF-Tu : CS molar ratio of 3.3 at
41 °C (Fig. 5A) and 6.7 a t 45 °C (Fig. 5B). Ovalbumin
(0.5 l
M
, Fig. 5A,B) and BSA (not shown) added to C S had
no protective effect on CS aggregation.
Recombinant pre-EF-Tu also protected MDH from
thermal aggregation. When heated at 45 °C, MDH began
to form insoluble aggregates, indicated by a n increase in
relative light scattering (Fig. 5C). Addition of various
amounts of pre-EF-Tu, however, reduced MDH
A
B
C
Fig. 2. Mass spectrometry analysis of rec om-
binant m aize pre-EF-Tu ( EF-Tu) isolated a nd
purified f rom E. coli expressing this protein.
(A) S core, number of matches, molecular
mass, and p I (isoelectric point) of p urified
protein identified by n an o-ESI-MS/MS. The
score was d eterm ined by the
PROTEINLYNX
1.1
Global Server (Microm ass) and is an indicato r
of search result quality. (B) Matching peptides
and a mino acid sequences of peptide ion
spectra obtained from t he trypsin digestion of
purified m aize pre-EF-Tu. (C) Matching sites
of peptide p roducts in the com plete sequence

of maize c hloroplast EF-Tu protein. T he
peptide products [ from (B)] are s hown in red,
blue, and gre en. The complete s eq uence was
obtained from the database using
PROTEIN-
LYNX
1.1 Global S erver.
Fig. 3. Binding of recombinant maize p re-EF-Tu (EF-Tu) to [
3
H]GDP.
Purified p re-EF-Tu was incubate d alone at 25 °Cor45 °Cfor45min.
Following i ncubatio n, t he activity of the p rote in was assessed by t he
[
3
H]GDP exchange assay [14]. Reaction mixture (total volume 200 lL)
contained binding buff er, 4.5 nmol of [
3
H]GDP (12.3 CiÆmmol
)1
), and
various a moun ts of EF-Tu. As controls, BSA and ovalbumin were
used. Reaction m ixtures were allowedtoequilibrateatroomtem-
perature for 10 min. Radioactivity was monitored using a B eckman
LS 6500 scintillation counter. Increase in radioactivity (DPM, disin-
tegration p er minute) indicates binding of pre-EF-Tu to [
3
H]GDP [14].
Binding of pre-EF-Tu to [
3
H]GDP suggests that this protein (pre-EF-

Tu) is p robably in a physiologically active form [14]. S imilar results
were obtained in a duplicate experiment.
Ó FEBS 2004 Chaperone activity of recombinant maize EF-Tu (Eur. J. Biochem. 271) 3687
aggregation and almost completely suppressed it a t a pre-
EF-Tu : MDH molar ratio of 10 (Fig. 5 C). O valbumin
(3 l
M
, Fig. 5 C) and BSA (not shown), in contrast, did not
protect MDH from thermal aggregation.
Recombinant maize pre-EF-Tu protected CS and MDH
from thermal inactivation
The recombinant maize pre-EF-Tu protected CS from
thermal inactivation. The enzymatic act ivity of CS was
severely halted when 2 l
M
CS was h eated at 4 3 °C alone or
in the p resence of e ither 4 l
M
BSA o r 4 l
M
ovalbumin; l ess
than 20 % o f the original CS activity r emained af ter 30 min
at 43 °C ( Fig. 6A). Upon temperature shift of the samples
to room temperature, the CS activity did not change
significantly, a s less than 20% of the original CS activity
remained after 75 min of recovery (Fig. 6A). In contrast,
when 2 l
M
CS was heated at 43 °C in the presence of 4 l
M

pre-EF-Tu, 46% of CS activity remained after 30 min
(Fig. 6 A). During the recovery period, the activity of C S
gradually increased, reaching a maximum of 68% of its
original activity after 45 min (Fig. 6 A). Similar results on
A
B
Fig. 4. Heat stability of purified recombinant maize pre-EF-Tu (EF-Tu).
(A) Relative ligh t sc attering of purified pre-EF-Tu du ring in cubation
at 45 °C.Aliquotofproteinsample(1 mL)wasincubatedat45 °Cfor
45 min, and light scattering was monitored at 320 nm. As a control,
heat-labile CS w as used. D ata represent averages of two independe nt
experiments. Bars indicate standard e rrors. N ote that during i ncu ba-
tion at 45 °C there is no increase in relative light scattering indicating
that the purified maize pre-EF-Tu is heat stable at this temperature. (B)
Western b lot of purified pre-EF-Tu. A sample of purified prote in
(1 mL ; 0.5 l
M
)wasincubatedat25°C (control) or 45 °C for 45 min.
Following incubation, samples were centrifuged and the supernatant
of each sample was analyzed for the presence of pre-EF-Tu using
Westernblottingandanti-myc Ig. Equal volumes of protein samples
were loaded in each lane. Note that t he pre-EF-Tu protein band is
present in the sample h eated at 45 °C, in dicating pre-EF-Tu stability at
this temperature.
Fig. 5. Effect o f recombinant maize p re-EF-Tu (EF-Tu) o n thermal
aggregation o f citrate synthase (CS; A and B) and malate dehydrogenase
(MDH; C). In separa te trials, CS a nd MDH were mixed with various
amounts of pre-EF-Tu . Three contro ls were used: C S or MD H alone,
CS or MDH mixed with ovalbumin, and C S or MDH mixed with BSA
(0.5 l

M
BSAwasmixedwithCSand3l
M
BSA was mixed with
MDH; not shown). Mixtures (to tal volume o f each m ixture, 1 mL)
were incubated a t in dicated tem per ature f or 45 min. During incuba-
tion, samples were monitored for their absorbance a t 3 20 nm, which is
indicative of l ight scattering due t o C S or MDH aggregation [ 20,21].
Data represent averages of two inde pend ent experiments. Bars indicate
standard errors. Note that pre-EF-Tu p rotected CS an d MDH from
thermal a ggregation. Note: BSA and ovalbumin wer e chosen as con-
trol proteins because they are k nown to b e r elatively heat stable a nd
have been used in chaperone (protein aggregation) studies [40]. In
addition, our preliminary ligh t scattering expe rimen ts showed that
BSA and ovalb umin are stable at 45 °C, as no increase in light
scattering (indicative o f protein aggregation [20]) was d etected when
BSA or ovalbumin were heated at this te mpera ture for 45 min (not
shown).
3688 D. Rao et al.(Eur. J. Biochem. 271) Ó FEBS 2004
CS activity were obtained when this enzyme w as heated at
other high temperatures, 38 °C, 41 °C, and 45 °C (not
shown).
The recombinant pre-EF-Tu also protected MDH from
thermal inactivation. When 0.5 l
M
MDH was heated at
43 °C a lone or in the presence of either 2 l
M
BSA o r
2 l

M
ovalbumin, t he MDH activity was very low, le ss
than 1% of its o riginal a ctivity a fter 3 0 min (Fig. 6 B).
However, when MDH was heated in the p resence of 2 l
M
pre-EF-Tu, 5 0% of MDH activity remained immediately
after heating (Fig. 6B). During the recovery period, the
activity of MDH did not change significantly (Fig. 6 B). A
similar effect of pre-EF-Tu on MDH activity was seen
when MDH was incubated at 38 °C, 41 °C, and 45 °C
(not shown).
The recombinant pre-EF-Tu did not show an effect on
reactivation of heat-inactivated CS and MDH (Fig. 7).
When CS and MDH were heated at 43 °C, witho ut pre-EF-
Tu, t he activity of CS and MDH w as severely reduc ed, a nd
the addition of pre-EF-Tu after heating did not change their
activity during the recovery (Fig. 7).
Discussion
Elevation of ambient temperature ( heat shock or heat stress)
affects cell metabolism, causing changes in the rates of
biochemical reactions and injuries to cellular membranes
[26,27]. Moreover, increases in ambient temperature also
cause denaturation and aggregation o f most proteins [ 27],
but protein denaturation due to heat shock is reversible
unless followed by aggregation [28].
Plants generally respond to high temperatures with the
induction of heat shock proteins ( HSPs). HSPs are t hought
to play a role i n heat tolerance by acting as molecular
chaperones; that is, they bind and stabilize o ther proteins,
protecting them from thermal aggregation a nd inactivation

(thermal damage) [29–31].
Recent studies have suggested that some protein synthesis
elongation factors may be involved in heat tolerance by
acting as molecular chaperones. Prokaryotic elongation
factors, EF-G [23] and EF-Tu [22], for example, interact
with unfolded and denatured p roteins, as do molecular
chaperones, and protect them from thermal aggregation.
Also, E. coli EF-Tu interacts p referentially with h ydropho-
bic regions of substrate proteins [32], a strategy used by
molecular chaperones to p revent thermal aggregation of
their substrate proteins [3 0].
Studies from our laboratory have implicated maize
EF-Tu in heat tolerance [5,9–11]. Maize EF-Tu exhibits
> 80% amino acid i de ntity w ith bacterial EF-Tu [5], and it
has been hypothesized that, in maize, this protein may show
chaperone activity similar to prokaryotic EF-Tu [5,9–11].
Fig. 6. Effect of recombinant m aize pre-EF-Tu ( EF-Tu) on the activity
of citra te synthase (CS; A) and malate de hydrogenase ( MDH; B) after
incubation at 43 °C. In separate trials, CS and MDH were mixed with
indicated amounts of recombinant pre-EF-Tu. Reaction mixtures
(total volume of each mixture , 1 m L) w ere incubated at 4 3 °Cfor
30 m in. After in cubation, th e mixtures w ere qu ickly c ooled to room
temperature and then kept on ice for up to 75 min (75 min recovery).
Where indicated, a mixture of CS and BSA or ovalbumin (ovalb.) was
used as control. CS and MDH activity was measured at various times
of rec overy. Data represent averages of three indepe ndent experiments
(standard e rrors are plotted but theyareoftensmallerthanthesym-
bols). Note that in the presence of recombinant pre-EF-Tu, CS and
MDH showed a r elatively high activity immediately after exposure t o
43 °C ( 0 m in of recovery). Similar results were obtained at 38 °C,

41 °Cand45°C (not shown).
Fig. 7. Effect of r ecombinant maize pre-EF-Tu (EF-Tu) o n the a c tivity
of h eat-inactivated CS and MDH. CS and M DH were incubated at
43 °C f or 30 min. Followin g incubation, indicated amounts of pre-EF-
Tu were add ed t o the he at ed prote in s amples, a nd th e r eaction m ix-
tures (total volume of each mixture, 1 mL) w ere allowed to re co ver on
ice for 45 min. The residual activity of CS and MDH w as measured at
room temperature a t v arious tim es o f recovery. Data represent aver-
ages of two independent experiments. Bars indi cate standard errors.
Ó FEBS 2004 Chaperone activity of recombinant maize EF-Tu (Eur. J. Biochem. 271) 3689
In this study, we isolated and purified the recombinant
precursor of m aize EF-Tu from E. coli and tested it for
possible chaperone activity. Before the chaperone studies
were cond ucted, the recombinant p re-EF-Tu was tested f or
its purity, identity, a bility t o bind GDP (indicative of EF-Tu
activity [14]), and the rmal stability. The re sults showed that
the recombinant pre-EF-Tu was isolated in h ighly pure
form (Fig. 1 ) capable of binding GDP (Fig. 3), and that the
identity of the protein, as determined by mass spectrometry,
was indeed maize EF-Tu (Fig. 2). In addition, the heat
stability tests showed that the recombinant pre-EF-Tu was
stable a t 45 °C ( Figs 3 and 4), t he highest temperature used
in our study to test th is protein for possible chaperone
activity. The thermal stability of m aize pre-EF-Tu obse rved
in our study was similar t o the thermal stability of bacterial
EF-Tu, which has been shown to b e s table a t t emperatures
ranging from 40 °Cto45°C[22].
Importantly, the recombinant maize pre-EF-Tu dis-
played chaperone activity. It protected the heat-labile
proteins, CS a nd MDH, from thermal a ggregation and

inactivation. The protective role of pre-EF-Tu against
thermal aggregation was exhibited in a concentration
dependent manner with t he most effective p rotection seen
when the molar ratio o f pre-EF-Tu : substrate p rotein (CS
or MDH) was 6.7 for CS and 10 for MDH.
The results on the influence of m aize pre-EF-Tu on
thermalaggregationofCSweresimilartothosereportedfor
bacterial EF-Tu [22]. Bacterial EF-Tu w as also found t o
protect CS from thermal aggregation in a concentration
dependent manner [22]. When 0.8 l
M
CS was heated at
43 °C it formed insoluble aggregates [22]. However, the
addition of 2 l
M
bacterial EF-Tu partially reduced, and
5 l
M
EF-Tu completely suppressed, the thermal aggrega-
tion of CS [22]. Thus, the most effective b acterial EF-
Tu : CS molar ratio that suppressed CS aggregation was
6.25 [22], and this is similar to the maize pre-EF-Tu : CS
molar ratio (6.7 at 45 °C) observed in our study.
Bacterial EF-Tu has been found to facilitate refolding of
denatured proteins [ 22,33]. Kudlicki et al.[33]haveshown
that Therm us t hermophilus EF-Tu h as chaperone- like capa-
city to as sist in the refolding of denatured r hodanese. A lso,
Caldas et al . [22] have observed refolding of urea-denatured
CS in the p resence of E. coli EF-Tu. In c ontrast to bacterial
EF-Tu, however, recombinant maize pre-EF-Tu does not

seem to have an effect on renaturation of denatured
proteins. Rather, this protein appears t o be important in
protecting proteins from thermal d amage during exposure
to heat stress. As seen in our study, maize pre-EF-Tu helped
CS and MDH maintain a relatively high activity during h eat
stress (Fig. 6) but it had no effect on r eactivation o f t hese
enzymes f ollowing their almost complete thermal inactiva-
tion (Fig. 7).
The ability of r ecombinant maize pre-EF-Tu to protect
model substrates (CS and MDH) from thermal damage
provides evidence for the possible role of native EF-Tu in
heat tolerance. Native chloroplast EF-Tu is predominantly
localized in the chloroplast stroma [11], and it is h ighly
possible that during heat stress this protein may protect
chloroplast stromal proteins from thermal damage by
acting as a molecular c haperone. T his possibility is suppor-
ted by Momcilovic & Ristic [11] and Ristic et al .[10]
who found that chloroplast stromal proteins from maize
genotypes with hi gher levels of EF-Tu display greater heat
stability (lower t hermal aggregation) than chloroplast
stromal proteins from genotypes with lower levels of
EF-Tu. The above hypothesis is also corroborated by
studies which showed that wh ole chloroplasts from a high-
level EF-Tu maize line are more heat stable than whole
chloroplasts from a low-level EF-Tu line [34,35] (in these
previous studies, maize EF-Tu was referred to as a
45–46 kDa HSP because the identity of this protein was
not known until the report of Bhadula et a l.[5]).
One could argue that the chaperone activity observed in
our study may be a n attribute of p re-EF-Tu, because o f the

presence of chloroplast targeting sequence, and that the
native EF-Tu may n ot have chaperone properties. We do
not completely rule out this possibility, however, the
evidence supports the hypothesis that the native maize
EF-Tu displays chaperone activity. Like n ative chloroplast
EF-Tu [12], the recombinant pre-EF-Tu shows the ability to
bind GDP (Fig. 3), an indication that the targeting sequence
does not significantly affect the activity of this protein.
Furthermore, as stated earlier, the amino acid s equence of
native maize EF-Tu is highly similar to t hat of bacterial
EF-Tu [5], which i s known to display chaperone activity
[22]. In addition, a comparison of the predicted two-
dimensional (
SCRATCH
servers; />tools/scratch/) and three-dimensional [36] s tructure reveals
a striking s imilarity between the native m aize EF-Tu and i ts
precursor ( pre-EF-Tu) i mplying that the functional proper-
ties of the native EF-Tu and pre-EF-Tu are similar. The
hypothesis t hat the native maize EF-Tu acts as a c haperone
and p rotects c hloroplast proteins f rom thermal aggregation
is consistent with the lower thermal aggregation of chloro-
plast s tromal proteins in m aize genotypes with higher levels
of EF-Tu as outlined above.
Plant cells possess many structurally diverse chaperones
[30,37,38], some of which, the small heat shock proteins
(sHSPs), function in conjunction with other chaperones
[21,31]. A model has been proposed for t he activity of
sHSPs [31,39]. D uring high t emperature stress, sHSPs bind
substrate p roteins in a n A TP-independent manner, p re-
venting their aggregation and maintaining them in a state

competent f or subsequent ATP-dependent refolding, which
is facilitated by other chaperones (e.g. HSP70 system)
[21,31,39]. This model is supported by L ee & Vierling [ 31]
who demonstrated that the HSP70 system is required for
refolding of a sHSP18.1-bound firefly luciferase.
Some plant sHSPs, however, can facilitate r eactivation of
heat-inactivated proteins d uring recovery from stress with-
out the presence of o ther chaperones and ATP. Pea (Pisum
sativum L) HSP17.7 and HSP18.1, for example, minimally
protected C S activity a t 3 8 °C, but helped this enzyme
regain 65–70% of its original activity after 60 min of
recovery at 22 °C [20]. The reactivation activity o f HSP17.7
and HSP18.1, how ever, seemed to be limited to tempera-
tures b elow 4 0 °C, as these two sHSPs had no effect on CS
reactivation following CS exposure to 45 °C[20].
Recombinant maize pre-EF-Tu does not seem to com-
pletely fit t he model proposed for the function of sHSPs,
and it differs from pea HSP17.7 and HSP18.1 in s ome
aspects o f its chaperone activity. Maize pre-EF-Tu appears
to be effective in protecting heat-labile proteins from
thermal damage without a r equirement for the presence of
3690 D. Rao et al.(Eur. J. Biochem. 271) Ó FEBS 2004
other chaperones and ATP. As our in v itro experiments
showed, maize pre-EF-Tu not only protected CS and MDH
from thermal aggregation (Fig. 5) but also helped CS and
MDH maintain a relativ ely high activity immediately a fter
exposure to h eat stress at t emperature above 40 °C (Fig. 6).
We do not know, however, if and how maize pre-EF-Tu
and/or native EF-Tu may function as molecular chaperones
in vivo. Our obs ervation that recombinant maize pre-EF- Tu

acts independently in vitro, without a requirement for o ther
chaperones and ATP, does not rule out the possibility that
in vivo this protein and/or its n ative form may function in
cooperation with other chaperones. Further s tudies are
needed to investigate this possibility.
In conclusion, in this study we demonstrate t hat, in vitro,
the recombinant maize pre-EF-Tu acts as a molecular
chaperone and protects heat-labile proteins, CS a nd MDH,
from thermal aggregation and inactivation. To o ur know-
ledge, th is is the first observation of chaperone activity by a
plant/eukaryotic precursor of the EF-Tu protein. Previous
studies have shown t hat whole chloroplasts [34,35] and
chloroplast s tromal proteins [10,11] from maize with higher
levels of EF-Tu display greater heat stability (lower t hermal
aggregation) than whole chloroplasts and chloroplast
stromal proteins f rom maize with lower levels o f EF-Tu.
Combined, our current and previous studies [10,11,34,35]
strongly support the hypothesis that maize EF-Tu plays a
role in heat tolerance by acting as a molecular chaperone
and protecting chloroplast stromal proteins from thermal
damage.
Acknowledgements
We acknowledge fi nancial support for this research from the United
States Department of Agriculture grant (Agreement no. 99-35100-8559)
to Z. Ristic. The authors are thankful to Drs Karen L. Koster and
Gary D. Small, The University of South Dakota, Dr Thomas E.
Elthon, The University of Nebraska – L incoln, and Dr David P.
Horwath, the U .S. Department of A griculture Experimental Research
Laboratory, Fargo, N D for c ritical reading of the manuscript.
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