From the Department of Biochemistry and the
§ Molecular Biophysics Unit, Indian Institute of Science,
Bangalore 560 012, India
Received for publication, August 6, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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Virus-like particles (VLPs) serve as excellent
model systems to identify the pathways of virus assembly. To gain
insights into the assembly mechanisms of the Physalis
mottle tymovirus (PhMV), six interfacial residues, identified
based on the crystal structure of the native and recombinant capsids,
were targeted for mutagenesis. The Q37E, Y67A, R68Q, D83A, I123A, and
S145A mutants of the PhMV recombinant coat protein (rCP) expressed in Escherichia coli were soluble. However, except for the
S145A mutant, which assembled into VLPs similar to that of wild type
rCP capsids, all the other mutants failed to assemble into VLPs.
Furthermore, the purified Q37E, Y67A, R68Q, D83A, and I123A rCP mutants
existed essentially as partially folded monomers as revealed by sucrose density gradient analysis, circular dichroism, fluorescence, thermal, and urea denaturation studies. The rCP mutants locked into such conformations probably lack the structural signals/features that would
allow them to assemble into capsids. Thus, the mutation of residues
involved in inter-subunit interactions in PhMV disrupts both subunit
folding and particle assembly.
Physalis mottle tymovirus
(PhMV)1 is a small
spherical plant virus consisting of a single-stranded, plus-sense RNA
genome with a size of 6.67 kb (1). The RNA genome is encapsidated in a protein shell comprising 180 chemically identical coat protein (CP)
subunits (20,000 Da) arranged in icosahedral symmetry. Depending on the
bonding interactions, the subunits are designated as A, B, and C (2).
The A-type subunits form pentamers at the 5-fold, and the B and C
subunits form hexamers at the 3-fold icosahedral axes. The protein
subunits are held by strong protein-protein interactions.
Purified preparations of tymoviruses, including PhMV, consist of empty
capsids (top component) in addition to the full capsids (bottom
component) containing complete genomic RNA. An interesting feature of
tymoviruses is that empty capsids are also formed in vitro
when the intact virus is subjected to a freeze-thaw cycle or when
exposed to high temperatures, alkaline pH, or denaturants (3-6). The
presence of empty shells in vivo and in vitro
suggests that the information required for error-free assembly is
contained within the primary structure of protein subunits in these
viruses. It is difficult to isolate an assembly-competent PhMV CP
subunit under non-denaturing conditions from isolated viral capsids
that can be used for deciphering the assembly pathway. Hence, the CP gene of PhMV and a number of its deletion mutants were cloned and
expressed in Escherichia coli. Interestingly, the
bacterially expressed rCP could self-assemble into stable virus-like
particles (VLPs) (7). Furthermore, the deletion of 30 amino acid
residues or the addition of 41 amino acid residues at the N terminus of rCP did not affect the assembly into T = 3 capsids. Several
site-specific mutants were also studied. One of the mutations, His-69
to Ala, led to the formation of a 19.4 S subassembly intermediate (8). Recently, the crystal structures of native and recombinant capsids of
PhMV have been elucidated (9, 10). To gain further insights into the
assembly mechanism of this virus, a careful analysis of these crystal
structures was made in the present study to identify residues involved
only in inter-subunit interactions, and these residues were targeted
for mutagenesis. Gln-37, Tyr-67, Arg-68, Asp-83, Ile-123 and Ser-145
were mutated to Glu, Ala, Gln, Ala, Ala, and Ala respectively, with a
view to examine whether such mutations could lead to the formation of
assembly intermediates or monomers. The mutant rCPs were expressed in
E. coli and purified. The mutants were characterized by
sucrose density gradient analysis, electron microscopy, high pressure
liquid chromatography, circular dichroism (CD), and fluorescence
spectroscopy. Our results imply that Gln-37, Tyr-67, Arg-68, Asp-83,
and Ile-123 are not only important for assembly but also crucial for
the proper folding of the subunits, whereas the interactions with
Ser-145 are not important either for folding or assembly.
Materials--
[ Primers--
The following primers were used to obtain rCP
mutants by the PCR-based approach (-S, sense; -AS, antisense): Q37E-S,
5'-TCCCTTTTGAGTTTGAAG-3'; Q37E-AS, 5'-CTTCAAACTCAAAAGGGA-3';
Y67A-S, 5'-CGCCCCCGCCCGACATGC-3'; Y67A-AS, 5'-GCATGTCGGGCGGGGGCG-3';
R68Q-S, 5'-CCCCTACCAACATGCTCAG-3'; R68Q-AS, 5'-CTGAGCATGTTGGTAGGGG-3';
D83A-S, 5'-TCCAACTGCTCTTGCTGTC-3'; D83A-AS, 5'-GACAGCAAGAGCAGTTGGA-3';
I123A-S, 5'-CGGCGCCGCCTCAGCCGCC-3'; I123A-AS,
5'-GGCGGCTGAGGCGGCGCCG-3'; S145A-S, 5'-TTGAAAGACGCCGTGACCTAC-3'; and
S145A-AS, 5'-GTAGGTCACGGCGTCTTTCAA-3'.
Site-directed Mutagenesis--
The rCP mutants were constructed
by the PCR-based method using corresponding sets of mutant primers
(11). The wild type pRBCP (i.e. CP gene in pRSET-B vector)
was used as the template for PCR amplification with the sense and
antisense primers with 2.5 units of Deep Vent DNA polymerase under
optimal conditions. The PCR-amplified mixtures were treated with
DpnI (10 units) at 37 °C for 1 h to digest the
methylated template DNA and then transformed into E. coli
DH5 Overexpression and Purification of Wild Type and Mutant
rCPs--
The wild type and mutant constructs were transformed into
the BL21 (DE3) pLys S strain. 500 ml of Terrific broth containing 50 µg/ml ampicillin was inoculated with 1% of the preculture. After
4 h of growth, the cells were induced with 0.3 mM
isopropyl-1-thio-
The mutant rCPs that failed to assemble were purified by Ni-NTA
affinity chromatography. The cell pellet after the induction was
resuspended in 100 mM Tris HCl buffer (pH 6.8) containing 300 mM NaCl and 5% glycerol. The sample was frozen at
Western Blot Analysis--
The mutant rCPs were separated on SDS
12% acrylamide gel and transferred onto nitrocellulose membranes (13).
The Western blot analysis was carried out using a monoclonal antibody
(PA3B2) specific for CP as the primary antibody and goat antimouse IgG conjugated to horseradish peroxidase as the secondary antibody as
described earlier (14). The blot was developed using diamino benzidine
in the presence of hydrogen peroxide in 0.05 M sodium citrate buffer (pH 4.8) containing trace amounts of cobalt chloride.
Gel Filtration Analysis--
The wild type and mutant rCPs were
analyzed on a Bio-Sil 400 gel filtration column using the Shimdzu LC6A
system. The column was equilibrated with the Tris-HCl buffer (pH 6.8)
containing 300 mM NaCl and 5% glycerol. The column was
calibrated with standard molecular weight markers supplied by
Bio-Rad.
Circular Dichroism--
CD spectra of protein samples (0.2 mg/ml) in 0.1 M Tris-HCl buffer (pH 6.8) containing 200 mM NaCl and 5% glycerol were recorded on a Jasco-715
spectropolarimeter (Japan Spectroscopic Co., Tokyo, Japan) at room
temperature (25 °C).
Thermal stability of the rCP and its mutants was assessed using CD by
following the changes in the secondary structure as a function of
temperature. The CD measurements were carried out in a Jasco-810
spectropolarimeter (Japan Spectroscopic Co., Tokyo, Japan) using a
bandwidth of 1 nm and a response time of 1 s in a cuvette with a
0.2-cm path length. The protein samples (0.2-0.3 mg/ml in 100 mM, Tris-HCl (pH 6.8) containing 200 mM NaCl
and 5% glycerol) were heated from 25-95 °C at the rate of
1 °C/min. The thermal unfolding was monitored at 222 nm, and the
ellipticity observed was used to calculate molar ellipticity ([ Fluorescence--
Fluorescence measurements were recorded on a
SPEX FluoroMax-3 Spectrofluorimeter in a 1-ml cuvette with a path
length of 1 cm at 25 °C. For measuring the intrinsic fluorescence,
the protein was excited at 280 nm, and the emission spectrum was
recorded between 300 and 400 nm. In the case of urea-induced
unfolding experiments, the fluorescence intensities were recorded at
324 nm, where the difference between partially folded (untreated) and 8 M urea treated proteins was maximum. The readings were
converted to relative fluorescence and plotted against urea
concentration. In all the experiments the protein concentration was 0.2 mg/ml in 0.1 M Tris-HCl buffer (pH 6.8) containing 200 mM NaCl and 5% glycerol at room temperature
(25 °C).
8-Anilino-1-naphthalenesulfonate (ANS) Binding--
ANS binding
was monitored by measuring the fluorescence intensities between 440 and
540 nm after excitation at 370 nm. In the case of urea unfolding, the
proteins (0.2 mg/ml) were incubated at various concentrations of urea
for 2 h at 25 °C. ANS was added to the protein samples to a
final concentration of 10 µM. The samples were excited at
370 nm, and ANS binding was monitored by measuring the fluorescence
emission at 470 nm. The readings were converted to relative
fluorescence and plotted against urea concentration.
Fluorescence Quenching by Acrylamide and Potassium
Iodide--
Fluorescence measurements of rCP and its mutants in the
presence of increasing concentrations of acrylamide/iodide (0-0.5M) were recorded on a SPEX FluoroMax-3 Spectrofluorimeter in a 1-ml cuvette with a path length of 1 cm at 25 °C. The fluorescence intensity was recorded at 324 nm after excitation at 280 nm. The fractional quenching (F0/F) was plotted against
acrylamide/iodide concentration (Stern-Volmer plot), where
F0 and F represent the fluorescence in the absence and
presence of acrylamide/iodide, respectively. In all experiments,
the protein concentration was 0.2 mg/ml in 0.1 M Tris-HCl
buffer (pH 6.8) containing 200 mM NaCl and 5% glycerol at
room temperature (25 °C).
Light Scattering Measurements of Urea Unfolded rCP--
Purified
rCP capsids were incubated with 0-8 M urea at room
temperature for 4-5 h. The absorbance was recorded at 325 nm.
Expression and Characterization of Site-specific Mutants of
rCP--
The site-specific mutants Q37E, Y67A, R68Q, D83A, I123A, and
S145A were generated by the PCR-based sense and antisense primer method
(11). The mutant clones were sequenced by Sanger's dideoxy chain
termination method to confirm the presence of the desired mutation
(15). The mutants were overexpressed in E. coli BL21 (DE3)
pLys S (16). All of the mutants were expressed to high levels (25-30%
of the total proteins) and were partially soluble. Approximately 60%
of I123A and S145A were present in the soluble fraction. The solubility
of the other mutant rCPs varied depending on the expression level in
different batches. On average, ~10-15% of the expressed protein was
present in a soluble fraction (data not shown). The mutant proteins
present in the soluble fractions were purified by the procedure similar
to that used for the purification of wild type rCP capsids (6). The rCP
expressed in E. coli has 41 additional amino acid residues
(including six histidines) at the N terminus and, as shown earlier (8),
it assembled into capsids that sedimented as a single peak when
subjected to sucrose density gradient centrifugation (fractions 12-16;
Fig. 1a). The S145A mutant rCP
co-sedimented with rCP capsids (Fig 1b). However, none of
the other mutant proteins exhibited peaks corresponding to assembled
particles (Fig. 1a). Furthermore, no assembled particles were seen when these mutant proteins were viewed under the electron microscope, whereas particles of 30 nm were observed for the mutant S145A capsids (Fig. 1b, inset). The mutant rCPs
that failed to assemble into T = 3 capsids could not be purified
by the procedures used for the purification of rCP capsids. Hence,
these mutant proteins were purified by Ni-NTA chromatography, making
use of the histidine tag at the N terminus. The purified proteins were analyzed by 12% SDS-polyacrylamide gel and were authenticated by
Western blot analysis (data not shown). The yields of the purified proteins were 10-15 mg/liter for Q37E, Y67A, R68Q, and D83A rCPs, whereas it was slightly higher (20-25 mg/liter) for the I123A rCP.
These purified proteins were also observed under an electron microscope, which further confirmed that these mutant rCPs failed to
assemble into virus-like particles. These purified mutants rCPs were
retained in the loading wells when run on 1% agarose gel and 8%
native polyacrylamide gels (data not shown), suggesting that these may
represent proteins that are folding defective.
Oligomeric Status of rCP Mutant Proteins--
Purified rCP capsids
and the mutant rCPs were loaded onto a Bio-Sil 400 gel filtration
column. The column was calibrated with standard protein molecular
weight markers. The rCP capsids eluted immediately after the void
volume as expected. However, the I123A mutant rCP behaved abnormally
and eluted after vitamin B12 (Fig. 2).
All of the three peaks in Fig. 2 were confirmed to be I123A mutant rCP
by Western blot analysis (data not shown). The Q37E, Y67A, R68Q, and
D83A mutant rCPs also behaved in a similar manner. S145A rCP capsids
co-eluted with rCP capsids (data not shown). Therefore, it is likely
that Q37E, Y67A, R68Q, D83A, and I123A rCP mutants exist as partially
folded monomers that bind to the column and are eluted later in the
run.
rCP Mutants (Q37E, Y67A, R68Q, D83A, and I123A) Exist in a
Partially Folded State--
Far UV CD was used to examine the
secondary structural features of the mutant rCPs that failed to
assemble into capsids. The far UV CD spectra of these mutants showed
troughs in the 208-225 nm range, suggesting that all of them had a
considerable amount of secondary structure (Fig.
3). Furthermore, the fluorescence emission spectra of these mutants showed an emission maxima at 332-336
nm and was similar to that observed with the rCP capsids (Fig.
4a). This indicated that the
aromatic residues, especially the single tryptophan residue, was
inaccessible to the solvent. Upon incubation with 8 M urea,
the emission maximum shifted to 350-356 nm, suggesting that the
tryptophan side chain was exposed to the solvent (Fig. 4b).
The fluorescence intensity was measured at 324 nm, at which the maximum
difference in intensity was observed between the native (0 M urea) and 8 M urea unfolded proteins. The
fluorescence intensities at various concentrations of urea were
calculated as a percent of the fluorescence intensity at 0 M urea. A discrete unfolding transition was found between 3 to 5 M urea in the case of rCP capsids (Fig.
5b), whereas no such transition was observed with the mutants rCPs (Fig. 5a).
This observation corroborates earlier studies in which the rCP capsids were found to be stable up to 4 M urea at room temperature
(7). Interestingly, the urea-induced denaturation of the rCP capsids, as measured by fluorescence, overlapped with the changes in light scattering as monitored by absorbance at 325 nm (Fig. 5b).
This indicated that disassembly and denaturation could be concerted events in this virus. The lack of a discrete unfolding transition in
the case of rCP mutants as monitored by fluorescence reflects conformational flexibility of these mutant proteins. Furthermore, the
absorbance at 325 nm for the mutant rCPs (0.2 mg/ml) was negligible (<0.005), suggesting that the spectral changes observed are not due to
aggregation.
The accessibility of tryptophan residues in the mutant rCPs
was further assessed by quenching experiments with acrylamide and
potassium iodide. Stern-Volmer plots for the wild type and mutant rCPs
(Fig. 6a) showed a similar
slope for Y67A, R68Q, and I123A mutant proteins, suggesting that the
tryptophan in these proteins are protected from the quencher to a
similar extent. The tryptophan in Q37E and D83A mutant rCPs, however,
seemed to be more accessible. Similarly, when potassium iodide was used as a quencher, subtle differences in the accessibility of tryptophan in
the mutants could be observed. Although rCP, I123A, and D83A had
similar slopes, Q37E, Y67A, and R68Q showed increased slopes (Fig.
6b).
Furthermore, the rCP mutants strongly bound ANS, suggesting the
presence of exposed hydrophobic pockets (Fig.
7a). ANS binding to rCP
capsids was less when compared with ANS binding to the rCP mutants
(Fig. 7a). This is understandable, as the capsids are held
by strong hydrophobic interactions and, hence, hydrophobic surfaces are
unavailable for ANS to bind. The urea-induced unfolding of the rCP
capsids and mutant rCPs was also monitored by measuring ANS
fluorescence. As in other globular proteins, there was an initial
increase (up to 3 M urea), followed by a decrease in ANS binding (up to 5 M urea) in the case of wild type rCP
capsids (Fig. 7b). The unfolding transition observed here is
in agreement with the results of urea-induced denaturation as monitored
by intrinsic fluorescence (Fig. 5a). In contrast, the mutant
rCPs showed only a continuous decrease in ANS fluorescence, suggesting that they were already in a partially unfolded state (Fig.
7b).
Thermal Stability of Mutant rCPs--
The partially folded nature
of the mutant rCPs was further assessed by monitoring the effect of
temperature on the molar ellipticity at 222 nm of the wild type and the
mutant rCPs. The wild type rCP and S145A capsids exhibited a
Tm of 88 and 83 °C, respectively (Fig.
8a). Q37E, Y67A, and R68Q
mutant rCPs did not show a discrete transition, whereas the D83A and
I123A mutant rCPs showed a broad transition between 50 and 70 °C
(Fig. 8b). These results further emphasize the
conformational flexibility in these mutant rCPs.
We have resorted to the molecular biology approach to decipher the
roles of specific amino acids in the formation of capsids of PhMV.
Earlier studies by site-directed mutagenesis have shown the presence of
a 19.4 S intermediate in the assembly/disassembly pathway (8). In this
study, we have carried out a structure-based mutagenesis of six
residues, namely Gln-37, Tyr-67, Arg-68, Asp-83, Ile-123, and Ser-145,
which are predominantly involved in inter-subunit interactions. Except
for S145A, all of the other mutant rCPs failed to assemble into T = 3 capsids, although they were found to be soluble (Fig. 1).
Furthermore, these mutant proteins are partially folded, as evidenced
by the following: (i) gel filtration elution behavior (Fig. 2); (ii)
the presence of secondary structure as shown by far UV CD (Fig. 3);
(iii) the lack of a sharp transition in thermal denaturation as shown
by CD (Fig. 8); (iv) persistent high levels of innate fluorescence due
to the single tryptophan present in the protein (Fig. 4); (v) lack of
discrete unfolding transition upon denaturation with urea (Fig. 5);
(vi) accessibility of tryptophan residue in the mutants as measured by
fluorescence quenching with acrylamide and potassium iodide (Fig. 6);
(vii) enhanced fluorescence of ANS in the presence of mutant proteins (Fig. 7); and (viii) the lack of a sharp structural transition in ANS
binding experiments in the presence of urea (Fig. 7). Thus, the
mutation of residues involved in extensive inter-subunit interactions affect both the subunit folding and particle assembly of PhMV. A
detailed analysis of the position of the residues mutated and the
probable consequence of the mutation on the folding and assembly is
presented below.
Gln-37 is conserved in all of the tymoviruses and is present at
hydrogen-bonding distance to the conserved glycines 113 and 114 and
leucine 109, respectively, at the A-A5
interface (Fig. 9 and Fig.
10a). Gln-37 also interacts
with Gly-113 and Leu-109 at B5-C interface and with Asn-188 at C-C2
contacts (Fig. 9 and Fig. 10a). This residue does not seem to interact
with any of the residues within the subunit. The mutation of Gln-37 to
Glu should therefore have disrupted only the inter-subunit
interactions. Replacement of Gln with Glu in the structure using the
molecular modeling software program "O" showed that there are no
short contacts and also that all the interactions of Gln-37 can be
retained by Glu. However, it introduces a negative charge in the dense
interface of subunits. The results presented here demonstrate that
the mutation of Gln-37, which is involved in multiple inter-subunit
interactions, also affects the proper folding of the subunit. Probably,
the negative charge introduced somehow interferes in the folding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP was obtained from
PerkinElmer Life Sciences. Restriction endonucleases and the
SequenaseTM Version 2 DNA sequencing kit were obtained from
Amersham Biosciences. Deep Vent DNA polymerase, DpnI, and
Ni-NTA agarose were obtained from New England Biolabs and Qiagen,
respectively. Ampicillin, isopropyl-1-thio-
-D-galactopyranoside, Coomassie
Brilliant Blue R-250, and ultra-pure urea were obtained from USB.
Diaminobenzidine, bromphenol blue, and EDTA were obtained from
Sigma. The oligonucleotide primers and conjugated double antibodies
were purchased from Bangalore Genei Pvt. Ltd., Bangalore, India. All
other chemicals were of analytical grade.
-competent cells. The plasmid DNA isolated from the colonies was
sequenced by Sanger's dideoxy chain termination method with a
SequenaseTM Version 2.0 DNA sequencing kit to confirm the
presence of the mutations.
-D-galactopyranoside for 4 h. The
virus-like particles formed (if any) were purified by using a procedure
similar to that used for purification of the virus (6). Briefly, the
cell pellet obtained after the induction was resuspended in 50 mM sodium citrate buffer (pH 5.5), sonicated, and the
supernatant was subjected to 10% polyethylene glycol precipitation.
The precipitate was resuspended in 50 mM citrate buffer (pH
5.5) and subjected to high speed pelleting. The pellet was dissolved in
citrate buffer and layered onto 10-40% sucrose gradient and
centrifuged at 26,000 rpm in an SW28 rotor for 3 h. The light
scattering zone was collected with a Pasteur pipette, diluted, and
subjected to ultra centrifugation for 3 h at 26,000 rpm. The
pellet obtained was resuspended in 50 mM sodium citrate
buffer (pH 5.5).
20 °C overnight, thawed at room temperature, and sonicated for 30 min. The sample was then centrifuged at 27,000 × g for
15 min to remove the insoluble debris. The soluble fraction was mixed
with 2 ml of Ni-NTA resin pre-equilibrated with the same buffer and
allowed to rotate in an end-to-end rocker for ~2 h. The protein resin
complex was washed extensively with 100 mM Tris-HCl (pH
6.8) containing 300 mM NaCl, 60 mM imidazole,
and 5% glycerol. The protein was eluted with 100 mM
Tris-HCl (pH 6.8) containing 300 mM NaCl, 250 mM imidazole, and 5% glycerol. The purity of the mutant
rCPs thus obtained was checked by 12% SDS-PAGE (12).
])
as seen in Equation 1,
where
(Eq. 1)
is measured ellipticity, Mr is
protein molecular weight, c is the concentration of the protein in
milligrams per milliliter, and l is the path length in centimeters.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sucrose gradient analysis of the recombinant
capsids. a, rCP and its mutants were layered onto a 10-40%
sucrose gradient and centrifuged at 26,000 rpm for 3 h using an SW
28 rotor. 1-ml fractions were collected from the bottom of the tubes,
and the absorbance at 280 nm was measured. rCP, Q37E, Y67A, R68Q, D83A
and I123A rCPs are shown as ,
,
,
,
, and
,
respectively. b, sucrose gradient analysis of rCP and S145A
capsids. rCP and S145A capsids mutants were layered onto a 10-40%
sucrose gradient and centrifuged at 26,000 rpm for 2.5 h using an
SW 28 rotor. 1-ml fractions were collected from the bottom of the
tubes, and the absorbance at 280 nm was measured. rCP and S145A rCP are
shown as
and
, respectively. The inset shows the
electron micrograph of S145A capsids.
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Fig. 2.
Size-exclusion chromatography of the rCP
mutant. The I123A mutant (upper
panel) was run on a Bio-SIL 400 column calibrated previously
with molecular mass markers (bottom panel). Peaks
a, b, c, d, and
e correspond to thyroblobulin (670 kDa), IgG (150 kDa),
ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12
(1.3 kDa) respectively. The top panel shows the elution
profile of I123A. The peaks 1, 2, and
3 obtained were authenticated by Western analysis. Similar
gel filtration profiles were obtained for the Y67A, R68Q, D83A, and
I123A mutants (data not shown).
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Fig. 3.
Far UV CD spectra of purified rCP and its
mutants. CD measurements were made using Jasco Spectropolarimeter.
The spectra were recorded between 200 and 280 nm at room temperature
(25 °C). rCP, Q37E, Y67A, R68Q, D83A, and I123A mutant rCPs are
shown as solid ( ), long dashed (
),
medium dashed (- - -), short
dashed (- - -), dotted (···), and
dash-dot-dash
(
·
) lines, respectively.
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Fig. 4.
Fluorescence emission spectra of rCP capsids
and its mutants. a, 0.2 mg/ml protein was excited at 280 nm,
and emission was monitored between 300 and 400 nm. rCP, Q37E, Y67A,
R68Q, D83A, and I123A are shown as solid ( ), long
dashed (
), medium dashed
(- - -), short dashed (- - -),
dotted (···), and
dash-dot-dash
(
·
) lines, respectively. b,
fluorescence emission spectra of rCP capsids and its mutants in the
presence of 8 M urea. 0.2 mg/ml protein was incubated in a
buffer containing 8 M urea for 2 h at room
temperature, excited at 280 nm, and emissions were monitored between
300 and 400 nm. rCP, Q37E, Y67A, R68Q, D83A and I123A rCP mutants are
shown as solid (
), long dashed (
),
medium dashed (- - -), short
dashed (- - -), dotted (···), and
dash-dot-dash
(
·
) lines, respectively.
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Fig. 5.
Urea denaturation studies of rCP and its
mutants. a, purified rCP mutants were treated with 0-8
M urea for 2 h at 25 °C. Protein was excited at 280 nm, and the emission was monitored at 324 nm. Fluorescence at 0 M urea concentration was taken as 100%, and the relative
fluorescence calculated was expressed as a function of urea
concentration. Q37E, Y67A, R68Q, D83A, and I123A mutant rCPs are shown
as ,
,
,
, and
, respectively. b, urea
denaturation of rCP capsids. Purified rCP capsids were treated with
0-8 M urea, and the fluorescence was monitored as
described above in panel a. Fluorescence emission of the
tryptophan at 324 nm (closed circles and
left axis) and light scattering measurements at
325 nm (open circles and right axis)
are indicated.
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Fig. 6.
Stern-Volmer plot of tryptophan fluorescence
quenching by acrylamide. a, purified rCP capsids and rCP
mutants were treated with 0-0.5 M acrylamide. The proteins
were excited at 280 nm, and the emission was recorded at 324 nm.
Fractional quenching F0/F is plotted as a function of
acrylamide concentration, where F0 and F represent the
fluorescence in the absence and presence of acrylamide, respectively.
rCP capsids, Q37E, Y67A, R68Q, D83A, and I123A mutant rCPs are shown as
,
,
,
,
, and
, respectively. b,
Stern-Volmer plot of tryptophan fluorescence quenching by iodide.
Purified rCP capsids and rCP mutants were treated with 0-0.5
M iodide. The proteins were excited at 280 nm, and emission
was recorded at 324 nm. Fractional quenching F0/F is
plotted as a function of acrylamide concentration, where F0
and F represent the fluorescence in the absence and presence of iodide,
respectively. rCP capsids, Q37E, Y67A, R68Q, D83A, and I123A mutant
rCPs are shown as
,
,
,
,
, and
, respectively.
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Fig. 7.
Fluorescence emission spectra of rCP capsids
and rCP mutants in the presence of ANS. a, ANS was added to
the purified mutants (0.2 mg/ml) from a stock solution of 1 mM to a final concentration of 10 µM. The
excitation wavelength was 370 nm, and fluorescence emissions were
measured between 440 and 540 nm. rCP, Q37E, Y67A, R68Q, D83A, and I123A
mutant rCPs are shown as solid ( ), dotted
(···), short dashed (- - -),
dash-dot-dot
(
··), medium dashed
(- - -), and
dash-dot-dash
(
·
) lines, respectively. b,
ANS binding to rCP capsids and rCP mutants in the presence of urea. The
mutant proteins were incubated in 0-8 M urea at 25 °C
for 2-3h. ANS was added to a final concentration of 10 µM. The samples were excited at 370 nm, and the
fluorescence emission was monitored at 470 nm. The readings were
converted to relative fluorescence and plotted against urea
concentration. rCP, Q37E, Y67A, R68Q, D83A, and I123A mutant rCPs are
shown as
,
,
,
,
, and
, respectively.
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Fig. 8.
Thermal denaturation of rCP capsids and its
mutants. a, Tm of wild type rCP
(line 1) and S145A rCP mutant (line 2)
are shown. Wild type rCP has a Tm of 88 °C,
whereas that of the S145A mutant rCP was found to be 83 °C.
b, thermal denaturation of rCP mutants. The thermal scans of
the Q37E (line 1), Y67A (line 2), R68Q
(line 3), D83A (line 4), and I123A (line
5) rCP mutants are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Coat protein subunits of PhMV. a,
ribbon diagram of the A, B, and C subunits of the PhMV coat protein in
the assymetric unit showing the mutations. The A, B, and C subunits are
colored green, red, and cyan
respectively. The residues selected for mutation are indicated.
b, diagram illustrating the convention used to designate
subunits on the T = 3 capsid of PhMV. The A subunits form pentamers at
the 5-fold axis, whereas the B and C subunits form hexamers at the
3-fold icosahedral axes.
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Fig. 10.
Interfacial interactions of PhMV.
a, interactions of Gln-37 (Q37) with residues
from the neighboring subunits. Hydrogen bonding interactions of Gln-37
with other residues at the A-A5, B5-C, and C-C2 contacts. The A, B, and
C subunits are colored in green, blue, and
red, respectively. b, interaction of Asp-83
(D83I) with Arg-159 (R159) from the neighboring
subunits. Asp-83 (D83) is involved in salt linkage with Arg
(R159) at the A-A5 and B5-C contacts. The A, B, and C
subunits are colored in green, blue, and
red, respectively. c, hydrophobic interaction of
Ile-123 (I123) with itself from the neighboring subunit at
the A-A5 contact. A subunits are shown in green.
d, interaction of Ser-145 (S145) with residues
from the neighboring subunits. Interactions of Ser-145 with other
residues at A-B, B-C, and C-A contacts are shown. The A, B, and C
subunits are colored in green, blue, and
red, respectively.
Arg-68, another conserved residue in all tymoviruses, is at hydrogen-bonding distance with Asp-136, Ser-137, and Val-138 at the A-B interface and is involved in a salt bridge with Asp-150 at the B-C interface (Fig. 9). This residue does not have any obvious intra-subunit interactions. The replacement of Arg with Gln can be done without short contact within the same subunit. However, its mutation to Gln led to the partially folded and assembly-incompetent protein. These results further support the observation that the mutation of residues involved in multiple inter-subunit interactions disrupts subunit folding and particle assembly. Even here, the uncompensated negative charge might interfere in the folding of the protein.
Asp-83 and Ile-123, although not conserved among all tymoviruses, are involved in extensive interactions with residues from the other subunits (Fig. 10, b and c). The mutation of these two residues to alanine also led to the partially folded proteins that were assembly-incompetent.
Tyr-67 is also conserved across the tymovirus group. It is involved not only in inter-subunit interactions at C-C2 and B5-A, but also in intra-subunit hydrogen-bonding interactions. Its mutation to alanine disrupts the hydrogen-bonding interaction at the quasi-2-fold axis. This creates a cavity in the vicinity of residues Leu-34, Leu-63, Leu-180, and Ile-32. This may have a destabilizing effect on the same subunit, which could hinder proper folding of the subunit. Hence, its mutation to Ala led to the misfolding of the subunits and the consequent arrest of assembly.
Interestingly, the tertiary structure of the partially folded mutant
proteins is similar to that of the rCP capsids (Figs. 4 and 6). The
single tryptophan residue, Trp-96, is located inside the -barrel,
and its mutation led to soluble aggregates defective in folding and
assembly (8). Furthermore, this Trp is not an interfacial residue.
Therefore, it is possible that the partially folded mutant rCPs and the
wild type rCP have similar accessibility to this tryptophan.
In contrast to the Q37E, Y67A, R68Q, D83A, and I123A mutant
rCPs, the S145A mutant assembled into capsids that were
indistinguishable from wild type rCP capsids. An analysis of the intact
PhMV capsid structure shows that Ser-145 has hydrogen-bonding
interactions with Thr-149 and Asp-150 in the A-B interface, Ser-19 and
Thr-149 at the A-C interface, and Asp-150 and Asn-139 at the B-C
interface (Fig. 10d). These interactions are presumably less
important or are compensated by other interactions in the mutant rCP
and thus do not affect the folding or assembly. Earlier studies on
D144N, N1881, and N188A mutant rCPs have shown that they do assemble into capsids, although these residues occur at the inter-subunit interfaces (8). Thus, none of the mutant proteins studied thus far has
resulted in a fully folded and assembly-competent monomeric subunit. It
is, however, possible that the mutant rCPs are not correctly folded
when synthesized in vivo and hence are assembly incompetent.
To examine this, the wild type rCP capsids were incubated with 8 M urea for 4 h at room temperature and subjected to
stepwise dialysis or immediate dilution (20-fold) to remove the
denaturant. However, it was not possible to obtain assembled particles
from such refolding mixtures. Invariably, the removal of denaturant led
to the complete precipitation of the protein. Because it was not
possible to refold and assemble the wild type rCP, similar experiments
with mutant rCPs were not attempted. In vivo studies on the
related turnip yellow mosaic virus has shown that the coat protein
synthesized in the endoplasmic reticulum accumulates in the cytoplasm
overlying clustered vesicles in the chloroplast (17). These become
inserted into the outer chloroplast membrane with the hydrophobic sides
that are normally buried in the complete virus particle within the
lipid bilayer. It is suggested that the nascent RNA strand emerges
through the vesicle neck and that virus assembly takes place in the
vesicular membrane (17). Thus, the partially folded CP attached
to the membrane, upon interaction with RNA, could further fold and
assemble into mature capsids. In the absence of RNA, these proteins
might self-assemble to form empty capsids. The results presented in
this communication clearly demonstrate that the mutation of residues
crucial for interfacial interactions disrupts subunit folding and
assembly in the PhMV. Furthermore, these mutants are partially folded
and are probably locked in conformations that lack the signals/features
that would allow them to assemble into T = 3 capsids. The
unfolding transition, as monitored by fluorescence, overlapped with the
changes in the light scattering when wild type rCP capsids were
denatured with urea, suggesting that disassembly and unfolding are also
concerted (Fig. 5b). Thus, there is a close connection
between folding and assembly in PhMV. In the flock house virus also,
the disassembly and unfolding of the mature capsids was shown to be
concerted (18). Viral structural proteins, such as the herpes simplex virus triplex forming protein (VP23), when expressed in E. coli, are shown to exist predominantly in a molten globule state
(19). The P22 coat protein has also been shown to be quite flexible in
solution (20). Thus, concerted coat protein folding and capsid assembly
may be a general phenomenon in viruses stabilized predominantly by
inter-subunit interactions.
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ACKNOWLEDGEMENTS |
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We thank Dr. V. Prakash, Dr. A. G. Appu Rao, Dr. Sridevi Annapurna Singh, and Mrs. Radha for help in carrying out thermal denaturation studies. We also thank Mr. Michael D. Silva and Ms. Eaazhisai for help in some of the experiments.
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FOOTNOTES |
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* This work was supported by the Departments of Science and Technology and Biotechnology, New Delhi, India.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. Tel.: 91-80-3601561 or 91-80-3942310; Fax: 91-80-3600814 or 91-80-3600683; E-mail: bchss@biochem.iisc.ernet.in.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M207992200
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ABBREVIATIONS |
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The abbreviations used are: PhMV, Physalis mottle tymovirus; CP, coat protein; rCP, recombinant coat protein; CD, circular dichroism; Ni-NTA, nickel-nitrilotriacetic acid; ANS, 8-anilino-1-naphthalenesulfonate; Tm, melting temperature.
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