From the
The highly conserved aspartic acid residue at position 87 of the
Escherichia coli chaperonin GroEL was mutated to glutamic
acid. When expressed in an E. coligroEL mutant
strain deficient for phage morphogenesis, plasmid-encoded GroEL mutant
D87E restored T
The GroEL protein of Escherichia coli is a member of
the sequence-related family of molecular chaperones termed chaperonins,
which are found in bacteria, chloroplasts, and mitochondria, and is
related to the TCP1 family in the cytosol of eukaryotes (see Hemmingsen
et al.(1988) and Lewis et al.(1992); for a review,
see Hendrick and Hartl(1993)). In the cell, GroEL and the co-chaperonin
GroES facilitate the folding of proteins during synthesis and transport
across membranes (Bochkareva et al., 1988; Kusukawa et
al., 1989; Cheng et al., 1989; Frydman et al.,
1994) and are involved in the protection of proteins from heat shock
(Kusukawa and Yura, 1988; Martin et al., 1992; Horwich et
al., 1993). In vitro, GroEL and GroES facilitate the
folding of a wide array of proteins (Goloubinoff et al., 1989;
Mendoza et al., 1991; Viitanen et al., 1992). The
GroEL oligomer is composed of two stacked rings of seven identical
57.3-kDa subunits with a cylindrical shape (Hendrix, 1979; Hohn et
al., 1979). GroES is a heptameric ring of 10-kDa subunits (Tilly
et al., 1981; Chandrasekhar et al., 1986) that can
bind one end (Saibil et al., 1991; Langer et al.,
1992) or both ends (Azem et al., 1994b; Schmidt et
al., 1994b; Llorca et al., 1994) of the GroEL
The GroEL
In this article, we
mutated the highly conserved aspartic acid residue at position 87 of
E. coli GroEL (Fig. 1) to glutamic acid. We found that
this mutation primarily affects the binding of nonnative proteins to
GroEL
In this work, the highly conserved residue Asp-87 of GroEL
chaperonins was mutated to glutamic acid. In a GroEL-deficient E.
coli host cell, plasmid-encoded mutant D87E restored phage
morphogenesis as efficiently as wild-type GroEL, but not the expression
of the lux operon, indicating that the mutant chaperonin is
partially functional in vivo. While under extreme conditions,
the D87E mutant may be less stable in vitro than wild-type
GroEL
The
spontaneous formation of the chaperonin-malate dehydrogenase binary
complex was four times less efficient for mutant D87E than for
wild-type GroEL
The binary complex between
mutant chaperonin and Rubisco was also less stable than the wild-type
binary complex when incubated for increasing periods of times with ATP.
Martin et al.(1991) suggested that ATP hydrolysis, in the
absence of GroES, can cause bound proteins to undergo multiple futile
cycles of release and rebinding to GroEL
A kinetic analysis of
ATP dependence curves of the GroEL ATPase showed that the maximal
ATPase activity of the mutant was 50% that of the wild type and that
the affinity of ATP for mutant D87E was an order of magnitude lower
than for wild-type GroEL
Higher concentrations of ATP
were required for the binding of GroES
A cluster of GroEL
mutants, all impaired in the ability to bind nonnative proteins, was
shown to be located in the apical domain of GroEL, facing the upper
central cavity of the oligomer (Braig et al., 1994; Fenton
et al., 1994). Remarkably, all the protein-binding mutants
were also found to be impaired in the ability to interact with
GroES
The K` and n
We thank Martin Kessel for providing the electron
micrograph in Fig. 2, S. Ulitzur for plasmid pChV1, G. Lorimer
for purified Rubisco, and S. Diamant and A. Azem for critical review
and discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
phage morphogenesis. It did not, however,
reactivate the transcription of a recombinant luciferase operon from
Vibrio fischeri. In vitro, GroEL mutant D87E was
found to be impaired in the ability to bind nonnative proteins and to
hydrolyze ATP, resulting in less efficient refolding of urea-denatured
ribulose-1,5-bisphosphate carboxylase/oxygenase. Mutant oligomer D87E
GroEL
was able to bind GroES
as efficiently as
wild-type GroEL
. The conserved aspartic acid residue at
position 87 located in the equatorial domain of GroEL (Braig, K.,
Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., Horwich, A.
L., and Sigler, P. B.(1994) Nature 371, 578-586) is thus
inferred to have a dual effect on the binding of nonnative proteins to
the GroEL
core chaperonin and on ATP hydrolysis.
cylinder.
oligomer can recognize and
spontaneously bind nonnative proteins (Goloubinoff et al.,
1989). The release of bound proteins in a folding-competent state
requires the hydrolysis of ATP and, under stringent conditions, the
presence of the co-chaperonin GroES
(Goloubinoff et
al., 1989; Schmidt et al., 1994a). The binding of ATP to
GroEL
and its subsequent hydrolysis occur with a high
degree of cooperativity (Gray and Fersht, 1991) and drive a
conformational change in the chaperonin molecule (Saibil et
al., 1993) that results in the release of the folding protein
(Martin et al., 1991). Mutant analysis, in conjunction with
x-ray structure analysis of GroEL (Fenton et al., 1994; Braig
et al., 1994), has provided a basis for understanding the role
of individual residues in specific chaperonin functions such as ATP
hydrolysis, binding of nonnative proteins, and binding of GroES. Thus,
it was inferred from two severe charge mutations that the aspartic acid
residue at position 87 of GroEL is located in the ATP-binding pocket.
Similarly, a cluster of GroES-binding and protein-binding double
mutants was identified in the apical domain of GroEL, facing the
central cavity of the GroEL
cylinder. This area was
therefore inferred to constitute a part of the protein-binding site of
the chaperonin (Fenton et al., 1994).
. It also affects ATP binding and hydrolysis, but not
the binding of GroES
.
Figure 1:
Evolutionary variability among
chaperonins. The amino acid sequence of GroEL from E. coli was
aligned in region 70-110 with 42 GroEL, cpn60, and hsp60
sequences from the SwissProt Data Base (1993) using the Pileup
algorithm (gap penalty of 3) in the 1991 Genetics Computer Group
software package (Devereux et al., 1984). Below the E.
coli sequence are listed the alternative amino acids found at each
position in at least one of the aligned sequences. Underlined residues are also conserved in chaperonins TCP1 and TF55 from
archaebacteria (Trent et al.,
1991).
Plasmid pTrcESL
The groEL gene was
amplified by the polymerase chain reaction from plasmid pKT200 (Bloom
et al., 1986) using DNA oligomers
5`-TGGTCGACAAAGACGTAAAATTCGGTA-3` and
5`-CCAAGCTTCTCGAGCTGGACGCACTCGC-3` that included the unique restriction
sites SalI and HindIII at the 3`- and 5`-ends of the
groEL gene, respectively. GroES was amplified from plasmid
pKT200 using the DNA oligomers 5`-GACCATGGAATTCCGTCCATTGCATGATCGC-3`
and 5`-GCGTCGACCATTATCTTTATTCCTTA-3`. The amplified DNA fragments
containing groES and groEL genes were introduced into
the multiple cloning site of pTrc99A (Pharmacia Biotech Inc.) and
called pTrcESL. The double mutant that resulted from the introduction
of a SalI restriction site at the N-terminal end of GroEL
(thus introducing the A to V and A to D mutations at positions 2 and 3
of GroEL, respectively) did not exhibit different in vivo behavior from the wild-type GroEL protein (Hemmingsen et al. 1988).
Mutagenesis
Site-directed mutagenesis was carried
out using the polymerase chain reaction (Higuchi, 1990) with pTrcESL as
a DNA template and the following two DNA oligomers:
5`-GCTGCAGGCGAGGGTACCACC-3` and 5`-GGTGGTACCCTCGCCTGCAGC-3`. The
resulting SalI-ClaI fragment, containing the D87E
mutation in GroEL, was introduced into pTrcESL, and the resulting
plasmid was called pTrc87. DNA sequencing of the entire groEL gene confirmed the D87E mutation.
Expression and Purification of GroEL and
GroES
Upon induction of the trc promoter of pTrc87 with
isopropyl-l-thio--D-galactopyranoside (40 µg/ml),
mutant D87E GroEL was overexpressed to a level at least 50-fold higher
than that of background GroEL levels of the E. coli host.
Purification of GroEL
was as described by Azem et al. (1994a) and that of GroES
as described by Todd et
al.(1993).
Electron Microscopy
Samples were applied to a
glow-discharged, carbon-coated, collodion-covered 300-mesh copper grid
and negatively stained with 1% aqueous uranyl acetate. Specimens were
viewed with a Philips CM12 electron microscope operating at 100 kV.
Micrographs were recorded on Kodak SO-163 emulsion at a nominal
magnification of 75,000.
ATPase Activity
The hydrolysis of ATP by
GroEL was measured as described by Azem et al. (1994a). GroEL
(3 µM protomer) was
incubated in 50 mM triethanolamine, pH 7.5, 10 mM
KCl, 10 mM MgAc
, and various concentrations of
[
-
P]ATP (0.01-5 mM with a
specific activity of 3 µCi/mmol) for 5 min at 37 °C.
Unhydrolyzed ATP was separated from the
P-labeled
inorganic phosphate by adsorption on 5% activated charcoal in 20
mM H
PO
according to Bais(1975). The
data for the velocity of the ATPase reaction were fitted to the Hill
equation using the nonlinear regression method of the Enzfitter
computerized program according to Leatherbarrow(1987). The following
constants were derived (see ): K
= V
/[GroEL], K`
= [S]
, and
n
= Hill coefficient.
Chemical Cross-linking of Chaperonin Oligomers
The
binding of GroES to wild-type and mutant GroEL
in solution was measured using chemical cross-linking at 37
°C with glutaraldehyde (0.22%) and SDS-polyacrylamide gel
electrophoresis as described by Azem et al. (1994a, 1994b).
Refolding of Rubisco
In vitro chaperonin-dependent refolding of Rubisco from Rhodospirillum
rubrum was assayed at 25 °C as described by Goloubinoff et
al.(1989). Rubisco (20 µM) was denatured in 4
M urea and 10 mM dithiothreitol and then diluted
80-fold into a solution of 50 mM Tris, pH 7.5, 20 mM
MgAc, 20 mM KCl, 1 mM dithiothreitol, 20
mM glucose, 1 mM ATP, 6 µM GroES
(protomer), and 3 µM wild-type or mutant GroEL. Hexokinase
(Sigma) was added to a final concentration of 40 µg/ml to stop the
refolding reaction at the indicated time points. Rubisco activity was
determined as described by Goloubinoff et al.(1989).
Inhibition of Protein Refolding
Pig heart
mitochondrial malate dehydrogenase (9 µM monomer;
Boehringer Mannheim), denatured in 4.5 M urea for 3 h at 25
°C, was diluted 70-fold into a refolding solution containing
increasing amounts of mutant or wild-type GroEL, 10
mM KCl, 20 mM MgAc
, 50 mM
triethanolamine, pH 7.5, and 2 mM dithiothreitol. Malate
dehydrogenase activity was assayed at 25 °C in 150 mM
K
phosphate buffer, pH 7.5, 0.5 mM
oxalacetate, 2 mM dithiothreitol, and 0.2 mg/ml NADH by
measuring the time-dependent change in the absorption of monochromatic
light at 340 nm (Miller et al., 1993).
Genetic Complementation of Phage Morphogenesis
The
E. coli mutant strain T carries a chromosomal
mutation in the groEL gene that prevents maturation of
-bacteriophage proheads (Takano and Kakefuda, 1972). T
was transformed with either pTrcESL or pTrc87, and phage
morphogenesis was tested using a ``spot test'' for plaque
formation with T
bacteriophage according to Revel(1980) and
Greener et al.(1993).
Genetic Complementation of Luciferase
Transcription
In E. coli, the transcription of a
plasmid-encoded luminescence (lux) operon from Vibrio
fischeri, pChV1 (Ulitzur and Kuhn, 1988), requires high levels of
GroEL and GroES (Adar et al., 1992). Wild-type E. coli cells carrying pChV1 emit levels of visible light 10 times higher than T
cells carrying pChV1 (Adar
et al., 1992). Cell lawns of T
carrying the
compatible plasmid pTrcESL or pTrc87 in addition to pChV1 were visually
tested in the dark for emission of light after a 20-h incubation at 30
°C.
Structure and Stability of Mutant D87E
To
analyze the effect of the mutation on the structure and stability of
the chaperonin oligomer, purified D87E GroEL molecules
were observed by negative stain electron microscopy and compared with
wild-type GroEL
(Fig. 2). Mutant D87E oligomers are
seen to be organized into characteristic tetradecameric double-layered
cylinders (Hendrix, 1979; Hohn et al., 1979), which are
indistinguishable from wild-type GroEL
. D87E displayed the
same mobility as wild-type GroEL
on nondenaturing 7%
polyacrylamide gels and was as stable as wild-type GroEL
in the presence of 1 M urea (data not shown). However,
at variance with the wild type, mutant GroEL
was half
dissociated into monomers in the presence of 2.25 M urea (data
not shown), suggesting that under extreme conditions, the mutant is
more prone to dissociation than the wild-type oligomer. Finally,
cross-linking with glutaraldehyde as described by Azem et al. (1994a) confirmed that within the time scale of the experiments
described, >95% of the mutant chaperonin was in the tetradecameric
form (data not shown). Thus, the general structure of the chaperonin
was not affected by the mutation.
Figure 2:
Electron micrographs of mutant D87E and
wild-type GroEL.
Genetic Complementation with Wild-type and Mutant
GroEL
In E. coli, expression of plasmid-encoded (pChV1)
luciferase operon from V. fischeri is under the
transcriptional control of GroE chaperonins (Adar et al.,
1992). Thus, wild-type E. coli cells harboring plasmid pChV1
emit 10-fold more visible light in the early
stationary phase than T
GroEL mutant cells. When E.
coli T
mutant cells harboring the two compatible
plasmids pChV1 and pTrc99A were spotted with T
bacteriophages, a plaque did not develop (Fig. 3A,
cell lawn 2) and the cell lawn emitted very low levels of
light (Fig. 3B, celllawn2).
When, however, the GroE-less pTrc99A plasmid was replaced by pTrcESL,
the T
cell lawn emitted high levels of light
(Fig. 3B, cell lawn 1) and developed a plaque
(Fig. 3A, celllawn1). When
pTrcESL was replaced by pTrc87 containing GroEL with the D87E mutation,
an intermediate phenotype was observed: the cell lawn emitted only low
levels of light (Fig. 3B, cell lawn 3) but
successfully developed a T
plaque (Fig. 3A,
celllawn3). Thus, the mutant chaperonin is
partially functional in vivo.
Figure 3:
Genetic complementation of a GroEL mutant
host strain. The E. coli GroEL mutant strain T (Takano and Kakefuda, 1972) was transformed with a plasmid,
pChV1, containing the luciferase operon from V. fischeri (Ulitzur and Kuhn, 1988) and with a compatible plasmid: wild-type
pTrcESL (celllawn1), GroE-less control
plasmid pTrc99A (celllawn2), or mutant
pTrc87 (celllawn3). Bacterial lawns on LB
agar plates were spotted with 3 µl of T
bacteriophage
(10
plaque-forming units/ml). After a 20-h incubation at 30
°C, the bacterial lawn was visualized for the formation of a
bacteriophage plaque in the light (A) and in the dark
(B).
Chaperonin-assisted Refolding of Rubisco in
Vitro
When compared with wild-type GroEL, the
chaperonin-dependent refolding of the Rubisco enzyme by mutant D87E
exhibited a slower rate and reached a maximum recovery of
25% that
obtained with the wild-type chaperonin (Fig. 4).
Figure 4:
Time course of Rubisco refolding by
wild-type and mutant D87E GroEL. Rubisco (20 µM),
denatured in 4 M urea, was diluted 80-fold into a refolding
mixture containing 6 µM GroES, 1 mM ATP, and 3
µM wild-type () or mutant D87E (
) GroEL as
described under ``Materials and Methods.'' Aliquots were
removed at various time intervals, and Rubisco activity was determined
as described by Goloubinoff et al. (1989). The percent
recovery is calculated relative to the activity of the same
concentration of native Rubisco.
Inhibition of Spontaneous Protein Refolding
At 25
°C, urea-denatured malate dehydrogenase can refold spontaneously
into native enzyme (Miller et al., 1993). In the presence of
GroEL, however, the malate dehydrogenase folding
intermediates bind to the chaperonin and are prevented from refolding
into an active enzyme. Fig. 5shows that the concentration of
D87E GroEL
required to inhibit half the spontaneous
refolding of malate dehydrogenase is four times higher than for
wild-type GroEL
. This indicates that the spontaneous
passive binding of nonnative protein is deficient in the mutant.
Figure 5:
Inhibition of spontaneous malate
dehydrogenase refolding by wild-type and mutant D87E GroEL. Malate
dehydrogenase (MDH; 9 µM) in 4.5 M urea
was diluted into a refolding solution containing increasing
concentrations of wild-type () or mutant D87E (
)
GroEL
. Malate dehydrogenase activity was measured as
described under ``Materials and Methods'' after 135 min of
refolding at 25 °C.
Stability of the Binary Complex
When wild-type
GroEL was first challenged with nonnative Rubisco,
incubated with ATP for increasing amounts of time, and then
supplemented with GroES
to initiate refolding, no
significant loss of recovered Rubisco was observed (Fig. 6,
). In contrast, when Rubisco-bound mutant D87E GroEL
was incubated with ATP for increasing amounts of time and then
supplemented with GroES
, a significant time-dependent loss
of recoverable Rubisco was observed (Fig. 6,
). If, however,
the initial incubation was carried out in the absence of ATP, no loss
of recoverable Rubisco activity was observed, either for wild-type or
mutant D87E GroEL
(Fig. 6,
and
,
respectively). These results further indicate that the binding and
rebinding of nonnative protein to the chaperonin are affected by the
D87E mutation.
Figure 6:
Rubisco recovery after incubation with or
without ATP. Urea-denatured Rubisco was diluted as described in the
legend of Fig. 4 in the presence of wild-type ( and
) or
mutant D87E (
and
) GroEL
. Binary complexes
were incubated for increasing amounts of time in the presence (
and
) or absence (
and
) of ATP prior to the
addition of GroES (6 µM). The GroES-dependent refolding
reaction was allowed to progress for 15 min and then was arrested with
glucose and hexokinase as in the legend of Fig. 4. The percent recovery
is calculated from the Rubisco activity recovered when ATP and GroES
were provided at the time of the Rubisco
dilution.
ATPase Activity
The rate of ATP hydrolysis by
mutant D87E is half that of wild-type GroEL (Fig. 7). summarizes the kinetic parameters
for the ATPase activity of mutant and wild-type GroEL
. The
affinity of ATP for mutant D87E was nearly an order of magnitude lower
than for wild-type GroEL
. Moreover, ATP was hydrolyzed by
the mutant in a noncooperative manner. However, GroES
inhibited ATP hydrolysis similarly in mutant and wild-type GroEL
(). This suggests that mutant D87E is deficient in the
ability to bind and hydrolyze ATP, but not in the ability to bind
GroES
.
Figure 7:
Time-dependent hydrolysis of ATP by
wild-type () and mutant (
) GroEL.
Binding of GroES
The binding of GroESto Mutant D87E and
Wild-type GroEL
to
mutant D87E or wild-type GroEL
oligomers in the presence
of ATP was measured using chemical cross-linking with glutaraldehyde
and SDS-polyacrylamide gel electrophoresis as described by Azem et
al. (1994b). In the presence of a saturating concentration of ATP,
GroES
has the same affinity for wild-type GroEL
(Fig. 8A) as for mutant D87E GroEL
(Fig. 8B). However, in the presence of a
saturating concentration of GroES
, GroES binding requires
5.7-fold less ATP to bind wild-type GroEL
(Fig. 8C) compared with mutant D87E
(Fig. 8D). The effective concentrations of ATP necessary
for the binding of GroES
to half of the GroEL
oligomers were 34 and 6 µM for mutant and wild-type
GroEL
, respectively. This suggests that functional binding
of GroES
to GroEL
is not directly affected by
the D87E mutation, but rather through the decreased affinity of the
mutant for ATP.
Figure 8:
Binding of GroES to
wild-type and mutant GroEL
. Wild-type GroEL
(3 µM protomer) (A) or D87E GroEL
(B) was incubated with 1 mM ATP and increasing
concentrations of GroES. The ratio between GroES and GroEL protomers
was 0, 0.125, 0.25, 0.50, 0.75, 1.0, and 2.0 in lanes
1-7, respectively. Wild-type GroEL (3 µM)
(C) or D87E GroEL (D) was incubated with an excess of
GroES (6 µM) and increasing concentrations of ATP (0, 1,
3, 5, 10, 50, 100, and 500 µM ATP in lanes
1-8, respectively). Cross-linking of chaperonin
hetero-oligomers with 0.22% glutaraldehyde, SDS-polyacrylamide gel
electrophoresis, and Coomassie Blue staining was according to Azem
et al. (1994a).
, it nevertheless assembled into a functional
oligomer with the same apparent structure as wild-type
GroEL
. Moreover, mutant chaperonin D87E retained, albeit
at various levels, all the chaperonin functions, thereby allowing an
analysis of the relationship between ATP hydrolysis, GroES binding,
protein binding, and protein folding in the oligomer.
, indicating that mutant D87E has a lower
affinity for nonnative proteins. Consistent with the 4-fold reduction
in the ability of the mutant chaperonin to bind nonnative malate
dehydrogenase, the maximal recovery of Rubisco by the mutant, in the
presence of ATP and GroES
, was
25% that of the
wild-type chaperonin (Fig. 4).
. While the
wild-type binary complex appeared to withstand multiple ATPase-driven
cycles of protein release and rebinding without a significant loss of
recoverable Rubisco, the mutant binary complex was significantly
destabilized by ATP hydrolysis (Fig. 5), suggesting that the
rebinding of Rubisco to the mutant is less successful than to the
wild-type molecule. This demonstrates that, although the mutation is in
the ATP-binding pocket, the ATP hydrolysis mechanism is functional and
is capable of driving reversible conformational changes in the mutant
chaperonin. Furthermore, it confirms that mutant D87E is primarily
affected in its ability to initially bind and to subsequently rebind
nonnative proteins during ATP-driven cycles.
. Fenton et al.(1994)
showed that two different charge mutations at the same position, D87K
and D87N, resulted in a complete loss of ATPase activity and,
consequently, of the protein refolding activity and GroES binding
ability. The effects of our D87E mutation as well as of D87K and D87N
are compatible with the structure analysis from x-ray crystallography,
which places Asp-87 in the ATP-binding pocket of GroEL (Braig et
al., 1994; Fenton et al., 1994; Kim et al.,
1994). The fact that a significant level of protein refolding was
achieved by a mutant lacking cooperativity in ATP hydrolysis suggests
that cooperativity of ATP hydrolysis may not be an absolute requirement
of the chaperonin-assisted protein folding mechanism, as previously
suggested (Bochkareva et al., 1992; Jackson et al.,
1993; Langer et al., 1992).
to the mutant
compared with the wild-type chaperonin oligomer (Fig. 8, C and D). This can be explained by the lower affinity of
mutant D87E for ATP. A titration of the GroES-dependent binding of
GroES
to GroEL
in the presence of a saturating
concentration of ATP showed that GroES
has the same
affinity for wild-type GroEL
as for mutant D87E
GroEL
(Fig. 8, A and B). This was
confirmed independently when the same concentration of GroES
inhibited half of the ATPase activity in the wild-type oligomer
compared with the mutant oligomer (). Furthermore, the
binding of GroES
to the mutant was functional since it
ultimately resulted in the refolding of Rubisco.
, suggesting that nonnative proteins and GroES
compete for common sites in the inner face of the apical domain
of the GroEL
core oligomer (Fenton et al., 1994).
Our findings suggest that the protein-binding sites are not necessarily
located in the apical region of the central cavity, but may be located
on the external envelope of the GroEL
cylinder, in the
equatorial domain of GroEL (Braig et al., 1994). This finding
is of particular importance in view of recent observations that
symmetric GroEL
(GroES
)
oligomers,
in which both access ways to the central cavity are obstructed, are
nevertheless fully functional chaperonins capable of successfully
assisting the refolding of nonnative proteins (Azem et al.,
1994b).
Table:
Characteristics of ATPase activity of wild-type
and mutant D87E GroEL
values for wild-type and mutant D87E GroEL were the average of
eight and six experiments, respectively. Values for the inhibition by
GroES of the GroEL ATPase were the average of four experiments.
K
values are from a representative experiment in
which the purified mutant D87E protein was devoid of detectable
degradation products as judged by native SDS gels (data not shown).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.