From the University of Connecticut, Department of Molecular and Cell Biology, Storrs, Connecticut 06269-3125
Received for publication, February 26, 2001
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ABSTRACT |
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Understanding the nature of protein grammar is
critical because amino acid substitutions in some proteins cause
misfolding and aggregation of the mutant protein resulting in a disease
state. Amino acid substitutions in phage P22 coat protein, known as
tsf (temperature-sensitive
folding) mutations, cause folding defects that result in
aggregation at high temperatures. We have isolated global
su (suppressor) amino acid substitutions that
alleviate the tsf phenotype in coat protein (Aramli,
L. A., and Teschke, C. M. (1999) J. Biol.
Chem. 274, 22217-22224). Unexpectedly, we found that a global
su amino acid substitution in tsf coat proteins made aggregation worse and that the tsf phenotype was
suppressed by increasing the rate of subunit assembly, thereby
decreasing the concentration of aggregation-prone folding intermediates.
The primary amino acid sequence of a polypeptide encodes all of
the information necessary for folding and assembly pathways, as well as
the native three-dimensional structure (2). Substitutions and deletions
in the amino acid sequence of a protein can have a significant impact
on the ability of a protein to fold or assemble properly. Depending on
the protein, such changes in the amino acid sequence can lead to
protein misfolding, mislocalization caused by misfolding, or
aggregation (3, 4). Amino acid substitutions in p53 lead to a
misfolding problem compromising the function of the protein, resulting
in cancer (5). Further, there are the diseases that result from
mislocalization caused by protein misfolding. For example, in
Although the process of protein folding is still not completely
understood, it is known that larger proteins often have identifiable folding intermediates. These folding intermediates may interact inappropriately before reaching the native state. In fact, protein misfolding is a common problem faced by biotechnology companies that
harvest proteins of commercial interest using recombinant DNA
technologies in heterologous hosts (7-11). Often these proteins have
problems with inclusion body formation, thereby decreasing the yield of
pharmaceutically important products. Single amino acid substitutions
can affect the folding pathway by shifting the folding from the
productive pathway to off-pathway aggregation. For example, the amino
acid substitutions in transthyretin causes a shift in the equilibrium
between the native state and an aggregation-prone unfolding
intermediate, resulting in amyloid formation. Individuals with any of
the 50 known amino acid substitutions in transthyretin are predisposed
to familial amyloidosis (12-15). During the folding of P22 tailspike
proteins with tsf
(temperature-sensitive folding) amino acid substitutions, a folding intermediate is aggregation-prone at high temperatures (16-19). Other proteins such as interluekin-1 We use coat protein of bacteriophage P22 as a model system to study the
processes of folding and assembly in vivo and in
vitro (22). P22 is a double-stranded DNA bacteriophage of
Salmonella typhimurium. The T=7 icosahedral capsid is
composed primarily of 420 coat protein subunits, each of which is a
47-kDa polypeptide of 430 amino acids. During the process of assembly,
the monomeric coat protein subunits interact with 150-300 molecules of
scaffolding protein (33 kDa) in a nucleation-dependent
reaction to produce the procapsid, a precursor of the mature capsid.
The nucleation-limited assembly reaction occurs by the addition of the
monomeric coat protein subunits to the growing edge of the partially
formed procapsid (23). Once the procapsid has assembled, the
scaffolding protein exits through the holes present in the procapsid
lattice while the DNA is actively packaged through the portal vertex.
During this process there is an expansion of the capsid lattice into the mature capsid, which is characterized by a 15% increase in diameter, a change in shape from the spherical to icosahedral, and the
partial closing of holes in the lattice (24-26).
Previously, a group of amino acid substitutions in phage P22 coat
protein that result in a tsf phenotype were identified and characterized (27, 28). In vivo, the tsf amino
acid substitutions significantly reduce the yield of soluble coat
protein at high temperatures because the newly synthesized
tsf coat polypeptides aggregate to form inclusion bodies
prior to reaching the mature assembly-competent state required for
capsid assembly (27, 29). As a consequence of the tsf
defect, there is a decrease in the rate and yield of procapsid assembly
both in vitro and in vivo (29, 30). In
vitro, tsf coat protein monomers have altered secondary
and tertiary structure, as well as increased surface hydrophobicity
(30). Additionally, the tsf amino acid substitutions cause
an increase in the rate of unfolding of coat protein, thereby increasing the concentrations of the folding
intermediates.1 These
in vitro properties may explain the increased propensity of
the folding intermediates to aggregate.
Based on the above observations, we present a model of folding and
assembly of coat protein (Fig. 1). It has
been established that the folding of coat protein proceeds through at
least two intermediates ([I1] and [I2]) to
form an assembly-competent coat protein subunit (30). In tsf
coat proteins, at high temperatures, there is an accumulation of an
aggregation-prone, off-pathway intermediate ([I*]) from either
[I1] or [I2] (30). The reaction producing
an aggregate is one of two essentially irreversible reactions in the
folding of coat protein. The second irreversible reaction is the
assembly of procapsids from the monomeric subunit, which occurs upon
the addition of scaffolding protein (31). The tsf coat
proteins assemble with slower kinetics than wild type
(WT)2 coat protein as a
result of the tsf folding defect (30) and assembly does not
correct the conformational defects of tsf coat proteins
(32).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin deficiency, a single amino acid
substitution results in the misfolding of
1-antitrypsin,
leading to the accumulation of long chain polymers within the
hepatocyte. This leads to a reduction of the plasma concentrations of
-antitrypsin and predisposes individuals to emphysema and liver
disease (6). Therefore, understanding the nature of protein grammar is
of paramount interest.
(20) and bovine growth hormone (21) have similar tendencies to aggregate.
View larger version (9K):
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Fig. 1.
A model of the folding and assembly of coat
protein. In our model, U represents the unfolded coat
protein, [I1] and
[I2] represent the intermediates,
[I*] represents an off-pathway intermediate that is
aggregation-prone, and N represents the folded state of coat
protein monomers and assembles into procapsids in association with
scaffolding protein. The irreversible reactions are indicated with the
heavy unidirectional arrows, whereas the reversible
reactions are shown with the lighter, bidirectional
arrows. The tsf amino acid substitutions increase the
rate of unfolding from the coat protein monomeric subunit to
[I2].1
As a means of identifying additional amino acids that are critical for folding, second site su (suppressor) amino acid substitutions of tsf coat protein mutants were isolated (1). The most frequently isolated type of second site suppressors were global suppressors. Global suppressor amino acid substitutions are capable of alleviating the phenotype of multiple tsf mutants. These global suppressors were identified at positions 163, 166, and 170 in the amino acid sequence of coat protein (1), a region located in close proximity of a putative hinge domain of coat protein (33).
Here we examine the mechanism by which a global suppressor alleviates
the tsf phenotype. Unexpectedly, we found that the presence of the global suppressor amino acid substitution T166I in the tsf coat proteins S223F and F353L lead to an increase in
aggregation. Further, we have determined that the increase in
aggregation of the tsf:su coat proteins is not likely to be
the result of a decrease in the thermostability of the
tsf:su coat proteins relative to the tsf parents.
However, by a novel mechanism, the tsf phenotype was
suppressed by an increase in the rate and yield of subunit assembly.
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EXPERIMENTAL PROCEDURES |
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Bacteria-- The bacteria used for all of the experiments were derivatives of S. typhimurium LT2. The amber suppressor minus host DB7136 (leuA414-am, hisC525-am) and its amber suppressor plus derivative DB7155 (leuA414-am, his C525-am, supE20-gln) have been described previously (34).
Bacteriophage-- The P22 bacteriophage used in this study were WT in gene 5, which encodes for coat protein, carried the tsf nucleotide substitutions in gene 5 leading to amino acid substitutions S223F or F353L, or carried tsf:su mutations in gene 5 leading to S223F/T166I or F353L/T166I (1). All phage strains used in these experiments carried the c1-7 allele, which prevents lysogeny. The phage also carried amber mutations in genes 3 and 13, to prevent DNA packaging and cell lysis, respectively, and to produce procapsids.
Chemicals-- Ultrapure guanidine hydrochloride, urea, and silver nitrate were purchased from Schwartz-Mann ICN. HGT Seakem agarose was purchased from American Bioanalytical. All other chemicals were reagent grade purchased from common sources.
Media-- LB was prepared as described by Life Technologies, Inc. and was used for initial bacterial growth of S. typhimurium, DB7155. A superbroth was prepared to support bacterial growth for procapsid isolation. The superbroth contained 32 g of tryptone, 20 g of yeast extract, 5 g of NaCl, and 5 ml of 1 N NaOH/liter of water (35).
Buffer-- The buffer utilized in all of the experiments was 20 mM sodium phosphate, pH 7.6. For procapsid preparations and storage of the shell stocks, the buffer utilized was 50 mM Tris base, 25 mM NaCl, and 2 mM EDTA, adjusted to pH 7.6 with HCl.
Purification of Coat Proteins--
WT, S223F, S223F/T166I,
F353L, and F353L/T166I used in the following experiments were obtained
from empty procapsid shell stocks that were prepared as previously
described (31, 32, 36, 37). Briefly, S. typhimurium (DB7155)
were grown in LB to 1 × 108 cells/ml at 30 °C and
infected with various strains of bacteriophage P22 at a multiplicity of
infection of 0.075 to prepare a large phage stock. To determine whether
reversions of the amber mutations or the tsf phenotype had
occurred, the phage stocks were checked for reversion frequency and
temperature sensitivity. Next, S. typhimurium (DB7136, an
amber minus strain) were grown in the superbroth to 4 × 108 cells/ml at 28 °C with vigorous aeration and
infected with bacteriophage P22 at a multiplicity of infection of 5. Because the phage carried amber mutations that prevent cell lysis and
DNA packaging, the infected cells accumulated procapsids. After 5 h the chilled cells were pelleted by centrifugation at 4 °C at
10,000 rpm using a GSA rotor in a Sorvall RC-5B Superspeed
centrifuge. The collected cells were suspended in a small volume of
cold buffer. The cells were lysed by a freeze/thaw cycle and were
treated with a final concentration of 0.1 M
phenylmethylsulfonyl fluoride and then with 50 µg/ml RNase and 50 µg/ml DNase. The procapsids were pelleted in a Ti60 rotor in a
Beckman L7-65 at 45,000 rpm for 35 min at 4 °C. The procapsid
pellet was resuspended in a small amount of buffer with fresh
phenylmethylsulfonyl fluoride by shaking overnight at 4 °C. The
procapsids were purified using a Sephacryl S-1000 (Amersham Pharmacia
Biotech) column to remove smaller contaminating proteins and membranes.
To prepare empty procapsid shells that are composed solely of coat
protein, the scaffolding protein was removed from the procapsids by
repeated extractions with 0.5 M guanidine hydrochloride
followed by centrifugation to pellet the shells. The shell stock
concentrations were determined by absorbance of the unfolded shell
stocks in 6 M guanidine hydrochloride using an extinction
coefficient of 0.957 ml mg1 cm
1 at 280 nm
(38). All purified empty procapsid shells were suspended in buffer and
stored indefinitely at 8.5 mg/ml at 4 °C.
Refolding of Coat Protein by Rapid Dilution-- Empty procapsid shells were unfolded at 2 mg/ml in 6.75 M urea, 20 mM phosphate buffer at pH 7.6 for 30 min at room temperature. Refolding was initiated by rapid dilution with phosphate buffer to yield a final coat protein concentration of 0.1 mg/ml with 0.34 M residual urea.
Refolding of Coat Protein by Dialysis-- Empty procapsid shells were unfolded in urea as described above. The denatured samples were diluted to 1.2 mg/ml and 4.0 M urea and then refolded by dialysis in a microdialyzer (Life Technologies, Inc.) at 4 °C at a rate of 0.75 ml/min with phosphate buffer. The refolded coat protein was collected when urea was no longer detected by refractometry. The samples were treated in a microcentrifuge for 2 min at 13,000 rpm at 4 °C. The protein concentration was determined by absorbance at 280 nm.
Native Polyacrylamide Gel and Agarose Electrophoresis-- The samples for native polyacrylamide gels were prepared by combining a portion of the protein with 3× native gel sample buffer (30% glycerol, 112 mM Tris, and 120 mM glycine). The samples (0.4 µg) were loaded onto native 4.3% polyacrylamide stacking gel (pH 8.3) and 7.5% native polyacrylamide resolving gel (pH 9.5) (39). Native polyacrylamide gels were run at 10 mA constant current at 4 °C for ~1 h. The bands on the native polyacrylamide gels were visualized by silver staining (40). The samples for the native agarose gel were prepared by combining a portion of the protein with agarose gel sample buffer (40 mM Tris base, 1 mM EDTA, 20% sucrose, pH 8.2, with acetic acid), and ~ 6 µg was loaded onto 1.2% Seakem HGT agarose gel made with the same buffer without the sucrose and run at 50 V for 3.5 h at room temperature (41). The agarose gels were stained with Coomassie Blue (10% acetic acid containing 0.03% Coomassie Brilliant Blue R-250 and 0.02% Coomassie Brilliant Blue G-250) overnight and destained (10% acetic acid and 10% isopropyl alcohol) over several days.
Procapsid Assembly Reactions--
To assemble coat protein into
procapsids, refolded coat protein at a final concentration of 0.83 mg/ml was mixed with scaffolding protein at a final concentration of
1.6 mg/ml at 20 °C in a total volume of 100 µl in the SLM
Aminco-Bowman 2 spectrofluorometer. The reaction was monitored by the
increase in 90° light scattering at 500 nm with the bandpasses set to
4 nm. Samples from each reaction were run on 1.2% agarose gels as
described above. Another method to monitor the assembly reaction is to
refold urea-denatured coat protein and urea-denatured scaffolding
protein together until the urea was no longer detectable by
refractometry (35). Coat protein and scaffolding protein were each
unfolded at 2 mg/ml in 6.75 M urea. The protein
concentrations for scaffolding protein were determined at 280 nm using
an extinction coefficient of 0.48 ml mg1
cm
1 (42). Samples were refolded together at 1 mg/ml in a
microdialyzer at a flow rate of 0.75 ml/min at 20 °C and 30 °C
until residual urea was no longer detected. The samples were prepared
and run on both native polyacrylamide and agarose gels as described above.
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RESULTS |
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As a means of identifying additional amino acids that are critical for folding, second site suppressors of P22 tsf coat protein mutants were isolated (1). The most frequently isolated group of second site suppressors were global suppressors at positions 163, 166, and 170 of the coat protein (1). Global suppressors have been identified in other proteins (43-45). For example, the global suppressors of the tsf mutants in P22 tailspike protein have been shown to alleviate folding defects by decreasing the rate of aggregation through stabilization of a folding intermediate (17, 46-48). Here we examine the mechanism by which a global suppressor alleviates the tsf phenotype in coat protein. Coat proteins carrying the global suppressor substitution T166I in the tsf backgrounds were chosen for this study because the temperature-sensitive phenotype of S223F and F353L was alleviated by all three global suppressors. Additionally, T166I was the most frequently isolated global suppressor amino acid substitution and has proven capable of improving the folding and assembly of tsf coat proteins to WT levels (1).
Aggregation of tsf and tsf:su Coat Proteins during
Folding--
Based on the results from the tailspike protein
experiments, we believed it likely that the global suppressors of coat
protein would also decrease the propensity of the protein to aggregate during folding. To determine the tendency of the tsf:su coat
proteins to aggregate during folding, WT, S223F, S223F/T166I, F353L,
and F353L/T166I coat proteins were first unfolded in denaturant.
Refolding was initiated by rapid dilution at various temperatures.
Aliquots were taken after refolding had been initiated and run on a
native polyacrylamide gel (Fig. 2). The
band of highest mobility corresponds to the folded monomeric form,
whereas the aggregates are the ladder of bands of slower mobility (11,
18, 30, 49). None of the coat protein samples aggregated at 15 °C or
below. The band of monomeric WT coat protein remained constant at all
temperatures tested. As previously reported, a small amount of
aggregation of WT coat protein was observed at 33 °C and above but
appeared to originate from a band of decreased mobility, suggesting
that the aggregation was likely due to incorrectly or slowly folding polypeptides that did not run in the monomer position on the native gel
(30). In contrast, the tsf mutant S223F began to
substantially aggregate at 20 °C. Surprisingly, S223F/T166I
demonstrated a significant increase in the rate of aggregation at
20 °C compared with its tsf parent, as seen by an
increase in the intensity and the number of bands of slower mobility
present in the tsf:su coat protein (Fig. 2). With the
increasing temperature, the intensity of the bands of slower mobility
further increased and the intensity of the monomeric bands decreased in
S223F/T166I over time when compared with its tsf parent,
S223F. The increased tendency to aggregate when the su
substitution T166I was present was also observed with F353L/T166I,
although both F353L and F353L/T166I began to aggregate at a higher
temperature than S223F and S223F/T166I. This is consistent with the
in vivo phenotype of F353L (1, 50). F353L began to aggregate
at 33 °C, whereas F353L/T166I had increased bands of aggregation at
the same temperature. Based on this experiment we conclude that the
presence of the su substitution T166I unexpectedly increased
the propensity for the tsf coat proteins to aggregate during
folding.
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Aggregation of tsf and tsf:su Coat Proteins from the Folded
State--
One possible explanation for why the tsf:su coat
proteins were more aggregation-prone was that these monomers were less
thermostable than the tsf coat protein monomers. Previously,
the folded tsf coat proteins were shown to be about as
stable as WT coat protein by differential scanning calorimetry and
denaturation with pressure (36, 51). However, recent equilibrium
folding and unfolding experiments with the tsf coat protein
mutants have indicated that the proteins were generally less stable to
denaturant than WT coat protein.1 Therefore, to determine
the thermostability of the tsf:su coat proteins, we first
refolded tsf and tsf:su coat proteins to their native conformation at 4 °C and then shifted samples to higher temperatures. The circular dichroism and tryptophan fluorescence spectra of the refolded tsf proteins were the same as
previously published (30). The spectra of the tsf:su
proteins were similar to their tsf parents and were
therefore consistent with folded structure (data not shown). Aliquots
were taken after the temperature shift-up and run on native
polyacrylamide gels to monitor aggregation of the proteins (Fig.
3). WT coat protein was resistant to
aggregation at the various temperatures tested up to 39 °C, where
aggregation begins, consistent with our previous work (30). The
S223F/T166I coat protein was only slightly more aggregation-prone than
the S223F coat protein. Similar results were obtained when comparing the thermostability of F353L and F353L/T166I, although they aggregated slightly less than S223F and S223F/T166I. Thus, it appears unlikely that the increase in aggregation of the tsf:su coat proteins
was due to a decrease in the thermostability of the folded monomeric state.
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Assembly of tsf and tsf:su Coat Proteins--
Since the
tsf:su coat proteins have an increased propensity to
aggregate during folding, the su amino acid substitution
must suppress the tsf phenotype at a step other than the
[I1 or I2] [I*] transition (Fig. 1).
Because procapsid assembly is the other irreversible step in the
folding pathway of coat protein and could potentially shift the folding
equilibrium to the right by decreasing the concentration of N, we
examined the effect of the su amino acid substitution on
this reaction. The samples of WT, S223F, S223F/T166I, F353L, and
F353L/T166I coat proteins were refolded by dialysis at 4 °C to form
monomeric coat proteins. The refolded coat proteins were mixed with
scaffolding protein at 20 °C. The assembly reaction was monitored by
the increase in light scattering at 500 nm (Fig.
4). WT coat protein was
assembly-competent and formed procapsids, as seen by the increase in
light scattering (31). The tsf coat protein S223F was
assembly-incompetent, and F353L had low assembly activity. In contrast,
addition of the su amino acid substitution in the
tsf background dramatically increased both the rate and the
yield of procapsid assembly. Because aggregates are large and can also
scatter light, we confirmed that the increase in light scattering was
the result of procapsid formation by running the assembly reactions on
native agarose gels where coat protein was found to be in either
procapsid form or as folded monomers (data not shown). Therefore,
whereas the su amino acid substitutions increased the
aggregation propensity of the tsf:su coat proteins during
folding, once folded, procapsid assembly was enhanced.
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In vivo, a P22 bacteriophage-infected cell would have
scaffolding protein present during the folding of coat protein. To
determine whether the presence of scaffolding protein during folding
would decrease the aggregation reaction by favoring subunit assembly, unfolded coat and unfolded scaffolding protein were mixed together at
equal concentrations and dialyzed at various temperatures until urea
was no longer detected (52). The samples were run on a native agarose
gel to detect procapsids (Fig.
5A). As expected, when the WT
coat protein was folded with WT scaffolding protein, subunit assembly
occurred, yielding procapsids. Conversely, the tsf coat
proteins S223F and F353L did not assemble into procapsids. The
tsf:su coat proteins S223F/T166I and F353L/T166I formed
procapsids at both 20 °C (Fig. 5A) and 30 °C (data not
shown), although there was a decrease in the yield of the procapsids at
the higher temperature. Additionally, the same samples were run on
native polyacrylamide gels to monitor aggregation (Fig. 5B).
We observed a significant decrease in the intensity of the bands
corresponding to aggregates in the reaction where the tsf:su
coat proteins were refolded with scaffolding protein as compared with
tsf:su coat protein refolded without scaffolding protein
(Fig. 5B). Thus, our experiments indicate that the presence
of scaffolding protein during folding of the tsf:su coat
proteins decreased their tendency for aggregation by increasing the
yield and rate of procapsid assembly.
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DISCUSSION |
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Aggregation is a serious problem both for biotechnology utilizing recombinant DNA technologies in heterologous hosts (7-11) and for various proteins with amino acid substitutions such as transthyretin (12-15). Thus, learning to control aggregation is of the utmost importance. Here we have investigated the effect of a global suppressor amino acid substitution on the aggregation propensity of tsf coat proteins in an effort to determine the mechanism of suppression of the tsf folding defects. Surprisingly, we have found that the tendency to aggregate increases in the presence of the suppressor amino acid substitution and that the suppression of the original tsf defect occurs by increasing the rate and yield of subunit assembly. This may have general implications in improving the folding and yield of multimeric proteins.
Global Suppressors--
In addition to our global suppressors of
tsf mutants in P22 coat protein (1), other second site
suppressors have been found that alleviate an original folding defect.
P22 tailspike protein is a well studied protein. A group of
tsf mutants of P22 tailspike protein have been identified
and characterized to be defective in folding and not stability (16, 53,
54). Analysis of the folding pathway revealed that aggregation of
tailspike protein occurs by the association of a partially folded
monomeric intermediate rather than the native trimeric species. The
thermolabile intermediate preferentially partitions onto the
aggregation pathway as the temperature increases (55). To determine
other positions in the amino acid sequence important to the folding
process, second site suppressors of the tsf tailspike
protein mutants were isolated (56). Two major global suppressor
substitutions were found to alleviate the folding defects of multiple
tsf mutants of tailspike protein (46). Structural resolution
of a truncated version of tailspike protein revealed that the two
global suppressors, V331A and A334V, were located in the parallel
-helix domain and the associated "dorsal fin domain" of the
fishlike structure. The global suppressors of the tsf
mutants in tailspike protein decrease aggregation in vivo
and in vitro (47). However, the global suppressor amino acid
substitutions act by different mechanisms (48). The suppressor, V331A,
alleviates the tsf folding defects by stabilizing the
completely folded protein by reducing steric hindrance in the native
state. The suppressor, A334V, alleviates the folding defects in a more
complicated way. The A334V suppressor substitution accelerates
unfolding at high temperature, thereby decreasing the stability of the
trimeric tailspike protein. The A334V substitution also increases the
stability of an early folding intermediate by improving hydrophobic
stacking during folding, therefore improving the overall folding
of the tailspike protein.
Examples of global suppressors of folding mutants of proteins
other than phage proteins exist. For instance, variants of
chloramphenicol acetyltransferase, a bacterial enzyme that confers
resistance to the antibiotic chloramphenicol, have been isolated. These
mutants quantitatively aggregate into cytoplasmic inclusion bodies,
resulting in a lack of chloramphenicol resistance. Van der Schueren
et al. (57) isolated second site suppressors of these
variants of chloramphenicol acetyltransferase. The global suppressor,
L145F, improved the thermostability of the protein and its ability to
fold into a soluble, enzymatically active conformation. Similarly,
temperature-sensitive mutants of the human receptor-like
protein-tyrosine phosphatase LAR have both stability and folding
defects that result in the aggregation of the protein. The presence of
a suppressor amino acid substitution in leukocyte-antigen related
(LAR) decreased the aggregation propensity as seen by an
increase in the production of the properly folded protein. The decrease
in aggregation occurs, at least in part, by an increase in the
stabilization of mutant protein (43). A second amino acid substitution,
M182T, is often found along with substitutions in TEM-1 -lactamase
that confer increased resistance to antibiotics (44). The M182T
substitution was shown to decrease the stability of the protein to
denaturant but increased the solubility of the double mutant (58). The exact mechanism by which M182T functions to decrease aggregation is
still to be established. Here the suppressor substitution has been
proposed to either inhibit aggregation by changing the conformation of
an aggregation-prone intermediate or to alter the folding mechanism in
a way to kinetically avoid aggregation (58).
Mechanisms for Suppression of Folding Defects--
Together, the
different suppressor amino acid substitutions of the various mutants of
P22 tailspike protein, chloramphenicol acetyltransferase, and human
receptor-like protein tyrosine phosphatase LAR have been shown
to correct the folding problem by stabilizing either the native
conformation of the protein or a folding intermediate. These examples
are in contrast to the novel mechanism we have elucidated for
tsf:su coat protein mutants. Based on our experiments, it is
clear that there is actually an increased propensity of the
tsf:su coat protein mutants to aggregate during folding.
However, the tsf:su mutant coat proteins have improved
subunit assembly capability, thereby suppressing the original
tsf phenotype. We believe that this method could be used
generally to improve the folding yield of multimeric proteins.
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ACKNOWLEDGEMENTS |
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We thank Carole Capen, Shannon Doyle, Dr. Todd Garabedian, Walter Nakonechny, and Mark Tardie for helpful comments and discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM53567.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.
To whom correspondence should be addressed: Dept. of Molecular and
Cell Biology, U-3125, University of Connecticut, 75 N. Eagleville Rd.,
Storrs, CT 06269-3125. Tel.: 860-486-4282; Fax: 860-486-4331;
E-mail: teschke@uconn.edu.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M101759200
1 C. M. Teschke, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: WT, wild type; LAR, leukocyte-antigen related.
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REFERENCES |
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