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J Biol Chem, Vol. 275, Issue 2, 1050-1056, January 14, 2000
From the Osmoprotectants exogenously supplied to a
hyperosmotic culture medium are efficiently imported and amassed by
stressed cells of Escherichia coli. In addition to their
evident role in the recovery and maintenance of osmotic balance, these
solutes should play an important role on the behavior of cellular
macromolecules, for example in the process of protein folding. Using a
random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition
of either lysine or osmoprotectants including glycine betaine (GB) in
the minimal medium. The growth rate increased proportionally with the
augmentation of the intracellular GB concentration. The mutation was
located in the lysA gene and resulted in the substitution
of the Ser at position 384 by Phe of the diaminopimelate decarboxylase
(DAPDC), which catalyzes the conversion of
meso-diaminopimelate to L-lysine. We purified
both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that
GB was capable of activating DAPDC-sf in vitro, thus
confirming the in vivo results. Most importantly, we showed
that the activation was correlated with a conformational change of
DAPDC-sf. Taken together, these results show, for the first time, that
GB may actively assist in vivo protein folding in a
chaperone-like manner.
Water availability is primordial for life of all organisms.
Bacteria submitted to a severe hyperosmotic stress instantaneously lose
a large amount of their intracellular water to balance the osmotic
strength between intracellular and extracellular spaces. The subsequent
decrease of cellular water activity together with the loss of cell
turgor lead to lessen the bacterial cell expansion rate (1). Surviving
such injuring conditions implies the reversion of water flux across the
cell membrane; this can be achieved by amassing highly soluble
compounds termed osmolytes (2, 3). Thus, Escherichia coli
cells rapidly take up high amounts of potassium ions (4, 5) and
subsequently increase their glutamate content to balance electric
charges. To avoid the perturbing effect of elevated ionic strength,
K+-glutamate can be progressively replaced by organic
osmolytes that behave neutral at physiological pH (6). Such compounds, termed compatible solutes (7), may be endogenously synthesized or
imported from the surrounding medium (3, 8). Imported compatible
solutes generally confer a high degree of osmotic tolerance to injured
cells. Among these so-called osmoprotectants, glycine betaine
(GB)1 is by far the most
effective and the most commonly assayed for hyperosmotic purposes.
In addition to the obvious predominant role they play in cellular
osmotic adjustment, internalized and accumulated osmoprotectants should
directly participate in other intracellular processes. Protective as
well as stabilizing effects of betaine and other solutes on proteins
denaturation because of increased salinity or temperature have been
reported (9-12). It is tempting to extrapolate these results in
vivo; however, bacteria submitted to elevated temperature do not
accumulate osmoprotectants. Similarly bacteria growing in high salinity
media and in the presence of low amounts of osmoprotectant accumulate
the latter at the expense of salts. Consequently in vitro
data concerning the beneficial effect of molar range of osmoprotectants
on the protection of enzymes against deleterious effects of salts and
temperature could not be extrapolated in vivo. Furthermore
we can question whether bacterial osmoprotectants are neutral, in
vivo, as commonly admitted (1), or whether they interact with
enzymes, inducing thereby structural and functional modifications.
Because in situ monitoring of enzyme activity is rather
difficult, this aspect has been scarcely studied. It was recently shown
that N-trimethylamine oxide induces in vitro
refolding of misfolded proteins (13, 14). It is therefore tempting to postulate that such effects may also occur in vivo with
osmoprotectants; hence, mutants unable to grow unless an osmoprotectant
is accumulated within the cell could be identified and used to develop
an osmoprotectant sensitive enzymatic probe.
In this report we describe the development of a lysA mutant
of E. coli, which grows only when glycine betaine is
intracellularly accumulated. An enzymatic probe derived from this
mutant was constructed, allowing us to ascertain that glycine betaine
induces, both in vitro and in vivo, the folding
of the modified enzyme. Thus glycine betaine is able to induce the
transition from an inactive to an active conformation of the enzyme,
thereby sharing properties somehow similar to those assigned to chaperones.
Bacterial Strains, Plasmids, and Growth Conditions--
The
E. coli strains and plasmids used in this study are listed
in Table I. Bacterial cells were grown
aerobically in LB medium or in M63 minimal medium with glucose or
lactose as carbon sources at 37 °C (15).
Mutagenesis--
Strain K10 was grown in liquid LB medium to
exponential phase (A570 = 0.8) and then washed
twice with citrate buffer (0.1 M, pH 5.5) prior to
mutagenesis with nitrosoguanidine as described (15). Mutants were
replica-plated on M63 containing or not 0.5 M NaCl and 1 mM GB. Colonies growing only in presence of NaCl and GB
were selected. Only a lysine auxotrophic mutant, N27, was retained for
this study.
Cloning and Nucleotide Sequence of lysA27--
All manipulations
with recombinant DNA were carried out according to standard procedures
(16) and the specifications of manufacturers. Chromosomal DNA of
strains K10 and N27 was digested with HindIII and ligated
with HindIII-cleaved pUN21. The ligation mixture was
transformed into JM1637 ( Construction of lysA Tagged Genes--
The 5' part of
lysA gene was amplified by polymerase chain reaction (PCR)
using pK10 as a template and using primers LysNdeI (5'-CCCCATATGCCACATTCACT-3') and LysEco3 (5'-CGCCTCTTCACCCTGTTG-3'). The resulting PCR product, possessing an engineered NdeI
site at the ATG start codon and the natural EcoRI site
within lysA, was digested with NdeI and
EcoRI and inserted between the corresponding sites of
pET22b+ yielding pET-LA5. This places the 5' part of lysA
gene, with its authentic ATG start codon, under the optimized transcriptional and translational signals of the vector. The 3' end of
lysA gene carrying or not the mutation was amplified by PCR
of pK10 or pN27 with the primers LysEco5 (5'-GCACATTGGTTCTGGCG-3') and
LysXho (5'-CCCCTCGAGCAATTCCAG-3'). The resulting PCR products possessed
the natural EcoRI site of lysA and an engineered
XhoI site just before the stop codon. The amplified DNA,
cleaved by EcoRI and XhoI, was inserted between
the EcoRI-XhoI sites of pET-LA5, giving pET-LA
(carrying the lysA) or pET-LA27 (carrying lysA
27) (see Fig. 1). These vectors contained lysA or
lysA27 fused to six contiguous histidine codon at the 3'
extremity; they are expressed from the T7 Diaminopimelate Decarboxylase Overproduction, Purification, and
Assay--
The BL21(DE3)/pET-LA or pET-LA27 strains were grown in 900 ml of LB medium supplemented with ampicilin (50 µg/liter). When the
A570 of the culture reached 0.6, the synthesis
of T7 RNA polymerase was induced by the addition of
isopropyl-1-thio- Size Exclusion Chromatography--
Size exclusion chromatography
was achieved by using two Superose 6 columns in series according to the
manufacturer's instruction (Amersham Pharmacia Biotech). Columns were
equilibrated with 40 mM Tris-HCl (pH 7.6), 200 mM NaCl-1 mM benzamidine and calibrated with
Glycine Betaine Accumulation and Diaminopimelate
Uptake--
[methyl-14C]GB (2 GBq mmol Tryptophan Fluorescence Emission Spectra--
Tryptophan
fluorescence emission spectra of the purified wild type diaminopimelate
decarboxylase (DAPDC) and the mutated enzyme (DAPDC-sf) were recorded
with an SLM-Aminco 8100 spectrofluorimeter operating in the ratio mode.
Excitation and emission slits were set to 4 nm. Emission spectra were
collected by using an excitation wavelength of 295 nm. All emission
data were corrected for instrumentation by using correction factors
provided by the supplier, and the buffer contribution was removed by
the proper emission subtraction.
Tryptophan Fluorescence Quenching--
Tryptophan fluorescence
quenching measurements were performed by using acrylamide, a
collisional quencher having an efficiency Dansyl Fluorescence Anisotropy--
The steady-state
fluorescence anisotropy of DAPDC labeled with the fluorescence probe
dansyl was investigated as described (27).
Infrared Spectroscopy--
Secondary structure analysis of
deuterated DAPDC and DAPDC-sf was carried out by Fourier transform
infrared spectroscopy of the amide I' band. IR spectra were collected
by using a Magna 460 from Nicolet equipped with a deuterated triglycine
sulfate detector. The samples were placed in a CaF2 cell
with a Teflon spacer of 0.05 mm. Band component analysis was achieved
by firstly estimating the number and position of elementary components
from the second derivative peaks in the 1600-1700 cm Isolation of Conditional Auxotrophic Mutants--
A
1-methyl-3-nitro-1-nitrosoguanidine mutagenesis was undertaken on
E. coli K10. Conditional auxotrophic mutants able to grow only in the presence of 0.5 M NaCl and 1 mM GB
were isolated. Among them, a stable lysine conditional auxotroph (N27)
required lysine for its growth in M63 medium. Alternatively, the growth of N27 was restored by the addition of 1 mM GB in M63
medium containing 0.5 M NaCl, 0.5 M KCl, or 0.9 M sucrose; these three media generate the same osmotic
strength of 1.2 osmol/kg H2O. However, in the presence of
the free permeant glycerol (0.9 M), GB was unable to
promote growth. Thus, growth restoration resulting from GB addition is
linked to osmolarity rather than to ionic strength or water activity.
When 1 mM of the osmoprotectants ectoine, proline, dimethylsulfonioacetate, or dimethylsulfoniopropionate were added to
0.5 M NaCl M63 medium, the growth rate of N27 was similar
to that observed in the presence of 1 mM GB. Hence the
reversion of N27 auxotrophy is not GB specific but seems to be
dependent on the presence of any osmoprotectant.
Identification of the Mutation--
Addition of diaminopimelate, a
biosynthetic precursor of bacterial lysine, to the medium was unable to
promote the growth of N27, although it was actively taken up by the
cells. Thus N27 seems affected in the last step of lysine biosynthesis,
i.e. in DAPDC activity. Indeed, the enzyme assay confirmed
that DAPDC activity was absent from the crude extract of N27. The phage
P1-mediated transduction experiment revealed 20% cotransduction
linkage of the mutation with argA, which suggests that the
mutation was located in the region of lysA-lysR (29).
Further complementation experiments of N27 by pLA17
(lysA+) or pLA40 (lysR+)
revealed that N27 has a mutation in lysA gene. The
corresponding allele was named lysA27. The wild type
lysA and the mutated lysA27 genes were cloned and
sequenced. The comparison of the two sequences allowed identification
of a single mutation corresponding to a C-T transversion in
lysA sequence, which induced the substitution of
Ser384 by Phe at the protein level. This mutated enzyme was
designated as DAPDC-sf.
Growth of lysA27 Strain Is Correlated to Intracellular GB
Level--
To study the dependence of lysA27 mutant upon
the intracellular GB level, the growth of the strains K10 and N27 was
followed in M63 medium of increasing salt content added or not with 1 mM GB (Fig. 2). In the
absence of GB, the growth rate of K10 slowed down when NaCl
concentration was increased and was abolished over 0.5 M
NaCl. The addition of 1 mM GB to the hyperosmotic medium allowed improvement of salt tolerance of bacterial cells. The intracellular GB concentration augmented in parallel to the NaCl content in growth medium. However, despite GB accumulation, the growth
rate of K10 decreased when the amount of salt was raised (Fig. 2).
The mutant N27 was unable to grow in the medium deprived of GB and
supplemented or not with NaCl. However, in the presence of GB, the
growth rate of N27 augmented proportionally with increasing concentration of NaCl that stimulates the uptake and intracellular accumulation of GB. Therefore, the growth rate of N27 was in direct proportion to intracellular GB content and became identical to that of
K10 at concentrations of NaCl higher than 0.5 M (Fig. 2).
Purification of the Mutated and the Wild Type DAPDC and in Vitro
Activation of the Mutated DAPDC--
The above mentioned results
indicate an in vivo activation of the mutated DAPDC. To
assess the activation mechanism, the wild type and mutated DAPDC were
purified. To facilitate the purification, a His6 codon tag
was introduced at the 3' extremity of the lysA and
lysA27 genes on the plasmids pK10-H6 and pN27-H6,
respectively. Growth parameters of the strains JM1637
(
DAPDC and DAPDC-sf proteins were purified to homogeneity. Analysis of
these proteins by size exclusion chromatography revealed that both
DAPDC and DAPDC-sf were located in fractions corresponding to the
molecular mass marker of 50 kDa. Because the molecular mass of DAPDC
deduced from lysA sequence is 46.177 kDa, the DAPDC must
function as a monomer. This result also rules out the possibility that
the loss of enzyme activity of DAPDC-sf would result from a failure of oligomerization.
The activity of purified DAPDC and DAPDC-sf were measured in
vitro in presence or absence of GB (Fig.
3). DAPDC activity was poorly affected by
increasing GB concentration. Its Km increased from 2 to 3.5 mM, and Vmax increased from
48 to 66 µmol of lysine formed/min/mg of protein in the presence of 1 M GB compared with that from medium deprived of betaine. In
contrast, GB exhibited a strong stimulating effect on DAPDC-sf
activity. This mutated protein was almost completely inactive in the
absence of GB, and its activity increased proportionally to GB
concentration in the assay medium (Fig. 3). In the absence of GB, the
Vmax value of DAPDC-sf was less than 0.01 µmol
of lysine formed/min/mg of protein, and its Km could
not be calculated. In the presence of 1 M GB the
Vmax increased to 4.4 µmol of lysine
formed/min/mg of protein, and the Km value was 62 mM. Although these values are much different from those of
the wild type enzyme, the 400-fold increase of
Vmax of the mutated DAPDC-sf is still significant. Therefore, GB is capable of activating the mutated DAPDC-sf, a result consistent with its capacity to restore the prototrophic phenotype of N27. The partial activation might result from
the lack of pyridoxal phosphate cofactor in the mutated DAPDC-sf as
observed by UV-visible absorption spectra analysis. Our attempt to
improve the in vitro activation by adding crude extracts in the reaction solution was not successful. Furthermore,
GB-dependent activation curves were the same for DAPDC-sf
present in the crude extracts of JM1637/pN27 or JM1637/pN27-H6 (data
not shown). Therefore, the addition of the His tag did not affect the
activation of DAPDC-sf by GB.
Structural and Dynamic Defects of DAPDC-sf--
To understand the
mechanism of GB-assisted in vivo activation of the mutated
DAPDC-sf, the relationship between the structure and activity of
DAPDC-sf was investigated in vitro by various approaches.
UV-visible absorption spectra of both DAPDC and DAPDC-sf molecules
(data not shown) indicated that the aromatic absorption band of
DAPDC-sf remained roughly unaltered compared with the one of DAPDC.
Upon selective excitation of tryptophan residues (Fig.
4), the fluorescence emission spectrum of
DAPDC-sf presented a total fluorescence intensity about 2-fold higher
than that of DAPDC, whereas a maximum emission wavelength at 328 nm was
observed for both enzymes. Such a
To probe whether these structural alterations have repercussions on
protein fluctuations, molecular dynamics was probed by collisional
quenching experiments in the presence of increasing acrylamide
concentrations. Fig. 6 displays the
Lehrer plots of DAPDC and DAPDC-sf. Linear regression of the data
yields similar accessible fractions of 1 for both DAPDC and DAPDC-sf
molecules. Assuming that each Trp residue has the same quantum yield
(which is likely because the Stern-Volmer plot yields a straight line), this finding allows to assess that all of the four Trp residues are
accessible to acrylamide in both the wild type and mutated enzymes.
However, the different slopes indicate that the quenching constant
K is largely altered as a consequence of the mutation. As
estimated from data, the DAPDC exhibits a quenching constant K of 3.51 M
To study the relation between activation and conformational change, the
effect of 1 M GB on the DAPDC-sf fluorescence emission spectrum was investigated (Fig. 4). The presence of GB drastically affects the Trp fluorescence of the DAPDC-sf in the sense that it
restores the emission spectrum observed with the DAPDC. Indeed 1 M GB induces a 2-fold decrease in total fluorescence
intensity without any Stokes shift variation. Thus, the fluorescence
emission properties of Trp in DAPDC and DAPDC-sf with 1 M
GB are very similar. A kinetic study of this transconformation has been
performed by monitoring both the GB-induced Trp fluorescence decrease
at 340 nm (Fig. 7) and the global protein
tumbling (data not shown) as a function of time. Data fitting of the
Trp emission decay leads to the conclusion that the transconformation
is best described by a single exponential; this implies that the
protein folding is ruled by a two-state equilibrium with relaxation
time Although in vitro studies showed for a long time that
osmoprotectants could influence protein stability and folding, such an
effect was never ascertained in vivo mainly because of the difficulty of monitoring such processes. To overcome the difficulty, we
have developed a genetic approach that allowed us to obtain osmoprotectants-dependent conditional auxotrophic mutants.
The DAPDC-sf of the mutant N27 contains a Ser-Phe substitution at the
amino acid 384, which results in total loss of the enzyme activity and
explains the lack of growth of this mutant in minimal medium in the
absence of lysine. Addition of osmoprotectants restores the growth of
this mutant, indicating the recovery of the DAPDC activity. We clearly
showed that the recovery of the activity of the mutated DAPDC is
proportional to the intracellular availability of glycine betaine.
These results suggest an in vivo osmoprotectant-assisted refolding of the DAPDC-sf. This hypothesis was further confirmed by
in vitro studies. GB significantly restores the activity of the purified DAPDC-sf, which is in parallel with refolding of the
mutated enzyme toward the conformation of the wild type enzyme as
monitored by various approaches.
The intrinsic Trp fluorescence emission spectra show that there are no
major changes in the solvent exposure of DAPDC-sf tryptophan residues.
These Trp residues may belong to hydrophobic nucleation domains, which
are not exposed to solvent either in DAPDC or in DAPDC-sf. Therefore,
the observed decrease of Trp quantum yield in DAPDC compared with
DAPDC-sf is best accounted for by a decrease in the Trp average
lifetime Glycine betaine is an osmoprotectant that is completely excluded from
the water hydration shell of proteins (9, 32). It has been thought that
this apparent high degree of exclusion was correlated with its action
as the most effective osmoprotectant. This clearly implies that this
perturbing solute does not bind to sites on the protein (33).
Increasing the osmolarity of the solution significantly reduces the
thermodynamic activity of water and thereby favors processes in which
water of hydration is displaced from biopolymer surfaces (33). The
DAPDC-sf folding here observed must necessarily favor protein-protein
interactions at the expense of water-protein interactions. One can
therefore conclude that the GB-induced DAPDC-sf folding process is
driven by a thermodynamic force because the presence of glycine betaine
drastically displaces the unfold-fold equilibrium toward the
conformation whose surface exposure to the solvent is reduced,
i.e. the folded species.
It can be proposed that glycine betaine like a chaperone assists the
folding of DAPDC-sf but probably in a different manner than does a
classical chaperone complex. The latter prevents hydrophobic aggregation by providing a hydrophilic environment (34-36), whereas the former forces the protein to decrease its exposure to solvent by
drastically decreasing the water activity (33). In vivo, chaperones and GB could exert a synergistic protection; nevertheless we
have shown that N27 (lysA27) growth strictly depends on GB (or other osmoprotectants) accumulation. So GB seems essential for the
folding of DAPDC-sf, whereas chaperones were unable to suppress the
deficiency of folding. Inefficiency of chaperones could be explained by
two hypotheses: (i) Chaperones do not interact with DAPDC-sf. This
protein proved to be stable and not submitted to proteolysis,
suggesting that it is not recognized as aberrant by the bacterial
proteases. Chaperones recognize the extended hydrophobic chains present
in loosely folded proteins (37, 38). Our results showed that such
hydrophobic domains are not exposed in DAPDC-sf. By contrast,
osmoprotectant efficiency does not rely on the recognition of a
particular protein domain. (ii) Chaperones are present in limiting
amounts in osmotically stressed cells. Chaperones are dramatically
overproduced in response to a temperature upshift (36) and, to a lesser
extent, to other stresses such as osmotic constraint in animal cells
(39, 40) and bacteria (41). Osmoprotectants are able to revert only the
osmotic induction of Hsp (40), probably because they are only
accumulated under osmotic stress; their accumulation in the absence of
an osmotic constraint would be pernicious for the cell. So, in a case
where osmoprotectants would accumulate in the cell, chaperones are
produced at a low amount, and the protection of destabilized proteins
is mainly ensured by osmoprotectants.
Why did two processes of protein protection arise during evolution?
Growth in high osmolality media increases the amount of misfolded
proteins. A large population of proteins interacting with chaperonins
for extended periods of time may be highly detrimental to the organism.
The capacity of glycine betaine to take part of the refolding task
allows chaperones to assist in protein synthesis and export, thus
maintaining an optimal growth rate. This phenomenon could exert a
strong influence on cellular metabolism, not only by maintaining normal
enzymatic activities but also by participating in the global regulation
of cellular metabolism when it concerns regulatory metabolic steps and
regulatory proteins. This latter point is of particular interest
because the search for a regulatory protein specifically involved in
osmoadaptation in E. coli remains unsuccessful (1). In
bacteria, osmoprotectants could participate in the global regulatory
processes by affecting the folding of central regulatory proteins as
RpoS (42) allowing a global regulation of metabolism without the
involvement of a specific regulator.
*
This work was supported by grants from the Centre National
de la Recherche Scientifique and the Direction de la Recherche et des
Etudes Doctorales.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
GB, glycine betaine;
PCR, polymerase chain reaction;
DAPDC, diaminopimelate decarboxylase;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.
Glycine Betaine-assisted Protein Folding in a lysA
Mutant of Escherichia coli*
,,,, and
Groupe Membranes et Osmorégulation,
CNRS UPRES-A 6026, Université de Rennes I, Campus de Beaulieu,
35042 Rennes, France, § Laboratoire de Biologie et Chimie
Moléculaires, Université de Bretagne-Sud, 12 avenue St
Symphorien, 56017 Vannes, France, and the ¶ Laboratoire de Chimie
Bactérienne, UPR 9043 CNRS, Institut de Biologie Structurale
et Microbiologie, 31 chemin Joseph Aiguier,
13402 Marseille, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bacterial strains and plasmids
lysA-lysR). Cells containing a
plasmid bearing the wild type lysA gene were selected on M63 minimal medium with ampicilin and tetracycline. Transformants containing a plasmid with the lysA27 allele were selected on
the same plates supplemented with 0.5 M NaCl and 1 mM GB. All transformants contained plasmids carrying an
identical 6.7-kilobase pair HindIII insert that covers the
lysA region of E. coli chromosome (17) (see Fig.
1). The lysA27 gene was isolated by subcloning a
2.3-kilobase pair HapI-HindIII fragment on
pBluescript SK
plasmid yielding pN27 (see Fig. 1). This
plasmid was able to complement JM1637 on M63-0.5 M NaCl-1
mM GB medium but not on M63 basal medium. The same
procedure allowed the recovery of the wild type lysA gene
yielding pK10 (see Fig. 1); this plasmid was able to complement JM1637
defect in M63 minimal medium. The nucleotide sequence of pK10 and pN27
inserts was determined by the dideoxy chain termination method
(18).
10 promoter in strain
BL21. The nucleotide sequence of pET-LA and pET-LA27 was confirmed. The
half-end of tagged genes were transferred from pET-LA to pK10 yielding
pK10-H6 and from pET-LA27 to pN27 creating pN27-H6 (Fig.
1). The tagged genes are transcribed
under the control of the lac promoter on these plasmids.
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Fig. 1.
Construction of plasmids bearing
lysA and lysA27. A,
genetic organization of the HindIII insert carrying
lysA (wild type) or lysA27 (mutant) genes in
pUN121. PCR products utilized for the construction of pET-LA and
pET-LA27 plasmids are drawn at the bottom of the restriction
map. B, structure of plasmids. Only the restriction sites
used are indicated in the figure. pK10 and pN27 plasmids were obtained
by subcloning 2.3-kilobase pair HapI-HindIII
fragment in SmaI-HindIII sites of pBluescript
SK . pET-LA and pET-LA27 plasmids were obtained as
described under "Experimental Procedures." These plasmids permit
the production of wild type and mutant DAPDC fused to the six-histidine
tag at the C-terminal part; the corresponding genes are transcribed
from 10 promoter. pK10-H6 and pN27-H6 plasmids were obtained by
substituting the 1.8-kilobase pair EcoRI-PvuI
fragment containing the 3' part of lysA by the corresponding
one of pET-LA and pET-LA27, respectively. These plasmids encoded tagged
DAPDC and DAPDC-sf; the corresponding genes are transcribed from the
lac promoter.
-D-galactopyranoside (final
concentration, 1 mM) and cells were grown for an additional 2 h at 37 °C. Proteins extraction and purification steps were performed at 4 °C using nickel-nitrilotriacetic acid-agarose column according to manufacturer recommendations (Qiagen). Both wild type and
mutated diaminopimelate decarboxylases were eluted at 150 mM imidazol. The proteins obtained contain only the
diaminopimelate decarboxylase when analyzed on SDS-polyacrylamide gel
electrophoresis that was performed according to Laemmli (19). Protein
concentration was determined according to Bradford (20).
Diaminopimelate decarboxylase assay was performed as described
(21).
-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum
albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.5 kDa). 200 µl of purified protein (about 2 mg of
protein) was applied onto the columns and were eluted at 0.2 ml/min
with the same buffer used for column equilibration. Fractions were collected, separated on SDS-polyacrylamide gel electrophoresis, and
analyzed by Coomassie Blue staining.
1)
was prepared from [methyl-14C]choline, 2 GBq
mmol1 (Amersham Pharmacia Biotech) according to Ikuta
et al. (22). The GB content of the cells grown in M63 medium
with various concentration of NaCl was determined in the presence of 1 mM [14C]GB as described previously (23).
meso-[14C-U]Diaminopimelate (12.9 GBq
mmol1; CEA, France) uptake was monitored as
for GB (23).
equal to unity (24). As
the DAPDC protein contains four tryptophanyl residues (positions 29, 140, 167, and 248), the Lehrer plot (25) was used to also determine the
tryptophan-accessible fraction to acrylamide. Data treatment was
essentially performed as described (26).
1
domain (28). Secondly, these parameters were introduced as first guess
for band curve fitting by nonlinear regression analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Influence of osmolarity and GB on growth
rate. K10 (circles) and N27 (squares)
strains were grown in M63 minimal medium at different NaCl
concentration in the absence (open symbols) or presence
(filled symbols) of 1 mM GB. Growth was measured
by monitoring A570 as a function of time.
Cellular accumulation of GB in both strains was measured in the
different growth conditions (triangles); it was identical
for the two strains.
lysA-lysR) containing pK10-H6 or pN27-H6 were similar to
those observed with K10 and N27 (Fig. 2) or with JM1673 carrying pK10
or pN27 (data not shown), respectively. Therefore, the addition of the
His6 tag at the C-terminal of wild type DAPDC or mutated
DAPDC-sf did not affect the enzymatic properties. The cellular content
of the two His tag enzymes was quantified after partial purification
using Ni2+-NTA agarose columns. The values obtained were
identical for the two strains in all growth conditions. These results
clearly showed that the restoration of the growth of N27 does not
result from an increase of enzyme synthesis but rather from the
activation, by GB, of the enzymatic activity of DAPDC-sf.
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Fig. 3.
In vitro effect of increasing GB
concentration on the activity of DAPDC (circles) and
DAPDC-sf (squares). The full 100% activity
corresponds to the activity of each enzyme with 1 M GB in
the reaction mixture, i.e. 66 and 4.4 µmol of lysine
formed/min/mg of protein for DAPDC and DAPDC-sf, respectively.
max of
fluorescence emission indicates either that the Trp residues are deeply
buried into the protein bulk (30) or that the indol rings interact with
hydrophobic residues. The large enhancement of Trp fluorescence in
DAPDC-sf may not arise from an altered exposure of Trp residues to the solvent. To assess this point, the secondary structures of DAPDC and
DAPDC-sf enzymes were investigated through infrared spectroscopy in the
amide I' band domain. DAPDC spectrum (Fig.
5) is composed of two major peaks located
at 1642 and 1652 cm1, which can be assigned to random
coil and -helix, respectively. The -helix component features 76%
of the total amide I' band area. The DAPDC-sf IR spectrum (Fig. 5) is
dominated by a large random coil component (1641 cm1),
which features as much as 84% of the total band area. A weaker band,
absent in DAPDC spectrum, is located at 1671 cm1 and may
feature -sheets or, more likely, turns and represents 16% of the
band area. It should be emphasized that, opposite to DAPDC, no helical
structure is apparent in DAPDC-sf. Hence, as a result of the mutation,
the DAPDC secondary structure shifts from a mainly helical protein to a
rather unordered protein displaying a low -sheet or turns
content.
View larger version (19K):
[in a new window]
Fig. 4.
Fluorescence emission spectra of DAPDC and
DAPDC-sf. Protein samples were diluted in 0.1 M
K/K2 phosphate buffer pH 6.8 for the fluorescence
experiments. Corrected emission spectra of proteins were collected at
the same A295, which is used as excitation
wavelength for the Trp fluorescence studies; they are the average of
three scans acquired with a 1-s integration time and a 1-nm step.
Protein concentrations were calculated on the basis of a molecular
extinction coefficient 
= 38,400 M1
cm1 at 280 nm, which derives from the Trp and Tyr protein
content. Fluorescence emission spectra were hence collected at the same
protein concentration from 300 to 400 nm. Solid line, DAPDC;
dashed line, DAPDC-sf; dashed-dotted line,
DAPDC-sf + 1 M GB.
View larger version (19K):
[in a new window]
Fig. 5.
Fourier transform infrared spectroscopy
spectra of the amide I' band of DAPDC and DAPDC-sf. Protein
concentration was 10 mg/ml. Spectra are the average of 128 scans
collected at a 2 cm 1 resolution. The buffer contribution
was interactively subtracted from the protein spectrum to yield a flat
base line in the 2000-1800 cm1 domain. The figure
displays the amide I' band contour of proteins along with the
underlying components resolved by band decomposition as described in
the text.
1 versus
2.08 M1 for the DAPDC-sf. This may indicate
that the protein fluctuations in the Trp environments are more
constrained in DAPDC-sf compared with DAPDC. As a whole, this in
vitro study clearly states that the loss of biological activity
arises from large structural and dynamic alterations of the enzyme,
which indicates that the mutated enzyme is not properly folded.
View larger version (17K):
[in a new window]
Fig. 6.
Dynamic quenching of DAPDC and DAPDC-sf by
acrylamide. The figure displays the Lehrer plot of the quenching
by acrylamide of DAPDC (circles) and DAPDC-sf
(squares). F0 and F were
the fluorescence intensities of the Trp residues in the absence and the
presence of acrylamide respectively; F = F 
F0.
= 9.8 min ± 0.7 (95% confidence interval). Dansyl
labeling experiments, which permit probing the global enzyme tumbling
through fluorescence anisotropy, further supports the proposed
mechanism of action of GB on the enzyme folding. The observed
anisotropies were 0.206 ± 0.007 (n = 15) for
DAPDC versus 0.1721 ± 0.001 (n = 15)
and 0.1474 ± 0.0017 (n = 15) for the DAPDC-sf in
the absence and in the presence of 0.5 M GB, respectively.
Clearly, these data indicate that the molecular shape of the wild type
and mutated proteins is different (they exhibit rotational correlation
time c of 16.5 and 10.9 ns, respectively) and that GB
addition results in a further decrease of c from 10.9 to
8.1 ns, which fits well with a folding process. Because dansyl
anisotropy is only sensitive to the global protein tumbling and, hence,
probe global events, attempts were made to correlate the dansyl
anisotropy decay to that of the Trp fluorescence emission previously
observed. Fitting of the data, once again, yields a single exponential
with a relaxation time = 10.1 min ± 1.9 (95% confidence
interval). This decay matches the one observed when probing Trp
emission intensity. In conclusion, it can be assessed that these
independent experiments actually reflect the same phenomenon,
i.e. the DAPDC-sf folding driven by GB. However, it should
be outlined that the rather low dansyl anisotropy value of DAPDC-sf
observed in the presence of GB as compared with DAPDC indicates that
although the Trp environments appear fully recovered, the molecular
shape (and hence the protein global conformation) of the mutated
protein still differs from that of the wild type; this finding well
agrees the partial restoration of the activity observed upon GB
addition.
View larger version (23K):
[in a new window]
Fig. 7.
Kinetics of folding of DAPDC-sf in 1 M glycine betaine at pH 6.8 and 20 °C.
Trp fluorescence intensity was monitored as a function of time
( exc = 295 nm; em = 340 nm). The data
reflect the intensity decrease upon GB addition (see Fig. 4). The data
are best fitted by a single exponential with a corresponding decay of 9.8 ± 0.7 min (95% confidence interval) as shown by the
linear behavior observed when the logarithm of the normalized
fluorescence intensity is plotted as a function of time (see
inset).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. This implies that the protein fluctuations around the Trp
residues (reflected by kq, the bimolecular collisional rate constant) are even larger in the DAPDC than in DAPDC-sf as deduced from quenching constants K. Indeed, the
observed quenching constant K is increased nearly 2-fold for
DAPDC as compared with DAPDC-sf. The constant kq,
which actually reflects the protein dynamics, must be accordingly much
more increased if 0 is decreased in DAPDC
(kq = K0). The most likely origin of this decrease in 0 must be a quenching of Trp
as a result of interactions with amino acid side chains. As a matter of
example, arginine is a potent Trp quencher. Because of the high
(~80%) helical content of DAPDC, it is likely that Trp residues are
located in -helices. Hence, Trp248 and
Arg252 must be in close contact (i+4) only if
the peptide backbone is helical; hence loss of this secondary structure
will separate Arg252 from Trp248 and in turn
eliminate any quenching effect of the guanidium group. This matches
well the drastic loss of helical content observed in DAPDC-sf as
compared with the DAPDC. This hypothesis is further supported by
secondary structure predictions, which indicate that the protein
sequence from 170 to 260 exhibits both a high hydropathic index and a
high helical probability with an hydrophilic gap around 238 (31).
FOOTNOTES
To whom correspondence should be addressed: Groupe Membranes
et Osmorégulation, CNRS UPRES-A 6026, Bâtiment 14, Campus
de Beaulieu, 35042 Rennes cedex, France. Tel.: 33-299-28-61-41; Fax: 33-299-28-61-40; E-mail: tbernard@univ-rennes1.fr.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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