From the Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294
Received for publication, August 23, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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The Salmonella typhimurium
bacteriophage P22 assembles an icosahedral capsid precursor called a
procapsid. The oligomeric portal protein ring, located at one vertex,
comprises the conduit for DNA entry and exit. In conjunction with the
DNA packaging enzymes, the portal ring is an integral component of a
nanoscale machine that pumps DNA into the phage head. Although the
portal vertex is assembled with high fidelity, the mechanism by which a
single portal complex is incorporated during procapsid assembly remains unknown. The assembly of bacteriophage P22 portal rings has
been characterized in vitro using a recombinant, His-tagged protein. Although the portal protein remained primarily unassembled within the cell, once purified, the highly soluble monomer assembled into rings at room temperature at high concentrations with a half time
of approximately 1 h. Circular dichroic analysis of the monomers and rings indicated that the protein gained The capsid proteins of many viruses are capable of self-assembling
into icosahedral structures with remarkable symmetry and fidelity.
During the morphogenesis of the double-strand DNA-containing phage, the
icosahedral symmetry of the head is interrupted by the presence of a
unique portal vertex. In all double-strand DNA phage characterized thus
far, this vertex is occupied by a single protein arranged as an
oligomeric ring (1-3). In each case, the portal rings have symmetries
that differ from the symmetry of the surrounding capsid resulting in a
symmetry mismatch that precludes a regular interaction between the
portal and capsid proteins (1). It has been suggested that this
symmetry mismatch allows for portal rotation during DNA packaging (1).
The formation of this vertex is essential for viability, because it
comprises a conduit through which the DNA enters and leaves the head
during the viral life cycle. Assembly intermediates during head
formation are short-lived and have not been characterized. This has
hampered efforts to characterize the mechanism of portal vertex
formation. The well-characterized assembly pathway of bacteriophage P22
is ideally suited for the continued investigation of such interactions.
During bacteriophage P22 morphogenesis, the earliest detectable
assembly intermediate is a procapsid (4, 5), which is composed of a
T = 7 icosahedral shell of coat protein surrounding a scaffolding
protein core (6), and ~12 molecules of the 84-kDa gene
1-encoded portal protein (7, 8). These three proteins (portal, coat, and scaffolding) are the only structural gene products required for head assembly and DNA packaging (9, 10). An enzyme complex
called a terminase binds to the portal vertex, and together, this DNA
translocation machine pumps the concatameric viral DNA into the
procapsid in an ATP-dependent manner (11-13). After the
head has been filled, the terminase complex cleaves the DNA releasing
the mature head (12). Mutants in the portal proteins of bacteriophages
SPP1 and P22 have been isolated that alter the packing density of the
DNA, suggesting that portal rings are part of the gauge regulating
packaging (14-16). Following packaging, the proteins of the tail
machine assemble at the portal vertex, stabilize the mature head, and
result in an infectious virion (17).
The ability of portal protein to form oligomeric rings without head
assembly has been reported for a number portal proteins studied thus
far, including those of phages T4, Cloning a His-tagged Portal--
Bacteriophage P22 gene
1 (portal) was amplified by
PCR1 from a wild-type phage
stock (c1 (25)). The amplified product was digested at
primer-encoded restriction sites, ligated into the pET-21b plasmid
(Novagen, Madison, WI), and transformed into Escherichia coli BL-21 (Novagen) for expression. DNA sequencing was used to verify that the recombinant portal gene contained the wild-type portal
coding sequence with a C-terminal addition of a Leu-Glu linker
(resulting from an XhoI site) and six consecutive histidines.
Construction of a Phage with His-tagged Portal--
A P22 phage
strain carrying the C-terminal His6-tagged portal protein
was generated by recombination. To allow for the efficient double-crossover needed to rescue a lethal point mutation in the phage
genome, the recombination template plasmid contained the C-terminal
His-tag sequence flanked on either side by ~1000 base pairs of DNA
complementary to the phage genome flanking the gene 1/gene
8 junction. The template was made by ligating two PCR
products that had been digested with AseI
(primer-encoded, on the right and left ends respectively): One
contained approximately the last 1000 base pairs of the His-tagged
portal gene and was generated from the expression plasmid described
above, the other contained ~1000 base pairs to the right of gene
1 and was generated from a wild-type phage stock. The
ligated template was gel-purified and ligated into the pCR-Blunt
plasmid (Invitrogen, Carlsbad, CA). To maintain the stop codon found in
the wild-type portal gene, a TAA codon was introduced in place of the
TGA codon found on the portal expression plasmid. In addition, the
AseI site used for ligating the recombination template
remained in the recombinant virus genome immediately after the gene
1 stop codon altering the wild-type gene 1/gene
8 junction.
This recombination plasmid was electroporated into Salmonella
typhimurium strain DB7000 (26) as previously described (27). Phage containing a cold-sensitive point mutation in the 5' region of
the portal gene (1
Purified phage containing the his-tagged portal were analyzed by
SDS-PAGE to determine the stoichiometries of the phage proteins and
compared with those of wild-type phage. No differences were detected.
The portal protein band in the purified phage was full-length and was
recognized by an anti-His-tag antibody. These phage were then examined
by negative-stain electron microscopy and found to be indistinguishable
from wild-type P22.
Protein Expression and Purification--
To generate soluble
protein, 500 ml of the expression host was grown at 37 °C under
ampicillin selection in 2× LB broth to an A600
of 0.7, induced with 1 mM
isopropyl-1-thio-
The cells were thawed at room temperature and two protease inhibitor
tablets were added (EDTA-free Mini Protease Inhibitor mixture, Roche
Molecular Biochemicals, Indianapolis, IN); the suspension was placed on
ice, allowed to lyse, sonicated to reduce viscosity, and centrifuged to
pellet material >~1000 S. Portal protein was isolated at 4 °C
from 25 ml of lysate by binding to a 5-ml nickel column (HiTrap
chelating, Amersham Pharmacia Biotech, Piscataway, NJ), washing with
~40 ml of 65 mM imidazole (in binding buffer), and
eluting with ~10 ml of 500 mM imidazole (in binding buffer). Portal protein stored in the elution buffer precipitated, and
EDTA was needed to maintain solubility at this step; therefore, the
protein was eluted into a tube containing enough 200 mM
EDTA such that the final EDTA concentration was ~10 mM.
The portal protein was usually contained in a 10- to 15-ml elution
volume at a concentration of 8-10 mg/ml. The entire process from
thawing to elution from the nickel column was usually accomplished
within 2 h.
Further purification was performed using anion-exchange and
size-exclusion chromatography (see "Results"). For long-term
storage, purified monomer was diluted to ~0.5 mg/ml and frozen in
aliquots at
Purified, recombinant portal protein was recognized by polyclonal
antibodies against wild-type portal protein, and also by anti-His4 antibodies (Qiagen). The purified monomeric
portal protein was analyzed using matrix-assisted laser desorption time
of flight mass spectroscopy to verify the molecular mass (83.6 kDa), and the integrity of the N- and C termini were verified by
tryptic and cyanogen bromide cleavage followed by mass spectroscopic
identification of the expected fragments (UAB Mass Spectroscopy Core Facility).
For use, the monomers were thawed on ice and concentrated (Macrosep 30, Pall Gelman Laboratory, Ann Arbor, MI followed by Centricon 30, Millipore, Bedford, MA) at 4 °C to minimize oligomerization. The
concentrated sample was injected onto a size-exclusion column (tandem
TSK6000/SW300), and the monomer peak was isolated into a tube sitting
in ice. Rings were prepared by allowing a sample of concentrated
monomer (50-100 mg/ml) to stand for 12-18 h at room temperature
(~23 °C). The protein was then diluted to ~1 mg/ml and allowed
to stand on ice for several hours. The ice equilibration step was
essential for preparing a stable homogeneous ring population, because
it apparently allowed time for unstable intermediates to dissociate to
the point that they were biochemically distinct from intact rings. The
rings were then purified using anion-exchange chromatography (see
"Results"), concentrated, and collected from the ring peak after
separation on a size-exclusion column.
Size-exclusion Chromatography--
For protein preparation,
portal monomer and rings were resolved using tandem TSK6000
PWXL (TosoHaas, Montgomeryville, PA) and 300SW (Waters,
Milford, MA) analytical size-exclusion columns running in 100 mM NaCl, 50 mM Tris-Cl, 2 mM EDTA
pH 7.4 at room temperature. For the kinetics experiments, the
populations were separated with only a 300SW to reduce run time. Data
were collected using the HPLC Manager program (Amersham Pharmacia
Biotech) running on a PC. Chromatogram baselines were adjusted using
the spectral analysis software Grams32 (Galactic Industries Corp.,
Salem, NH) and finally plotted using Origin 6.0 (Microcal, Northampton, MA).
Analytical Ultracentrifugation--
Equilibrium sedimentation
was performed using an XL-A analytical ultracentrifuge (Beckman
Coulter, Fullerton, CA). Data were collected at 11,000, 13,000, and
15,000 rpm at 1.2, 0.9, and 0.6 mg/ml at 4 °C. A partial specific
volume of 0.7288 for the portal protein was calculated from the primary
amino acid sequence using the protein analysis software SedNTerp
(University of New Hampshire, Durham, NH). The resulting concentration
profiles were fit with the program Origin 4.1 (Microcal) utilizing the
Beckman XL-A AutoR1 macro (Beckman Coulter), which allowed global
fitting of the absorbance profiles. The resulting data was then
exported and plotted using Origin 6.0 (Microcal).
Electron Microscopy--
Phage or portal rings were diluted to
~0.05 mg/ml, adhered to a carbon-coated Formvar layer supported on a
copper grid, blotted to remove excess material, fixed for 1 min with
0.5% formaldehyde, and stained for 20 s with a 2% solution of
uranyl acetate prepared in water. Images were collected on film with an
electron microscope (Hitachi Instruments, San Jose, CA) operating with
an accelerating voltage 75 kV.
Determination of the Activation Energy of Ring
Formation--
Portal monomer was concentrated at 4 °C using a
Centricon-30 (Millipore), the buffer was changed to 100 mM
NaCl, 50 mM NaHPO4, pH 7.4, using a spin column
(Micro Bio-Spin 6, Bio-Rad, Hercules, CA), and aliquoted into
thin-walled PCR tubes sitting on ice. To initiate polymerization, a
sample was placed in the preheated block of a thermal cycle machine and
incubated. At various times after the start of incubation, an aliquot
from the tube was sufficiently diluted in HPLC buffer to stop ring
formation (0.1 mg/ml), and the sample was analyzed using SEC (SW300).
The chromatographic peak of the rings overlapped that of intermediates,
which prevented a baseline separation of the ring peak for integration.
Therefore, to determine the contribution of rings to each chromatogram,
each chromatogram was computationally deconvolved using singular
value decomposition (SVD, (29, 30)). This was performed using an add-on
array-BASIC SVD program running in Grams32 as previously described
(31). The concentration of subunits in rings was determined by
calculating the relative fractional contribution of a homogeneous ring
chromatogram to each experimental chromatogram and then multiplying by
the total concentration of protein present in each sample. These values
were plotted versus time and fit to obtain relaxation times
( Circular Dichroism--
The CD spectra were collected using an
Aviv 62DS circular dichroism spectrophotometer (Aviv Instruments Inc.,
Lakewood, NJ). Far UV spectra from 200 to 260 nm were collected at a
protein concentration of 0.2 mg/ml in phosphate buffer (100 mM NaCl, 50 mM NaHPO4, pH 7.4) in a
0.1-cm path length cuvette at 25 °C. Data were collected at 0.5-nm
intervals as the average of five scans with 10-s averaging at each
wavelength. Under these conditions, high solvent absorbance prevented
the collection of data below 200 nm.
Data were averaged and smoothed using the on-board software, the
solvent background was subtracted, and the spectra were transferred to
a separate PC for further analysis. The amount of secondary structure
was estimated using the software program CDNN (version 2.1) (33). This
program used a trained neural net algorithm to compare the experimental
spectrum to a set of 33 reference spectra for which the secondary
structure content is known. The reliability of the estimations of
secondary structure content was determined by the closeness of the sum
of percentages to 100%.2
Thermal denaturation of the monomers and rings were performed in a
1.0-cm path length stirred cuvette in phosphate buffer. The CD signal
at 222 nm was collected in 0.5 °C steps with 20 s of averaging
per step. Two independent melting plots were obtained using different
preparations of monomer and ring. These were averaged to generate the
plot in Fig. 6.
Bis-ANS Fluorescence Measurements--
The fluorescent dye
4,4'-dianilino-1,1'-binapthyl-5,5'-disulfonic acid (bis-ANS, Molecular
Probes, Eugene, OR) was prepared as a stock solution in 50% methanol,
and the concentration was determined spectrophotometrically after
dilution into water (
Fitting of the reverse titration fluorescence data with the a
single-site hyperbolic-binding function allowed determination of
Fmax which represented the signal of bis-ANS
fully bound to the highest affinity site on the protein
(Fobs = (Fmax × L × Ka)/(1 + L × Ka)), where Fobs equals the
observed relative fluorescence signal above background,
Fmax is the maximum fluorescence signal of the
ligand complex, L is the ligand concentration, and
Ka is the apparent association constant (36).
Kd (1/Ka) was then determined by
calculating the free protein concentration during each measurement as a
function of fluorescence and once again fitting with the same binding
function (37).
Proteolytic Digestion--
Portal monomers and rings were
digested at 0.11 mg/ml with His-tagged Portal Protein Functions in Vivo--
Previous studies
had indicated that the C-terminal 50 amino acids of the P22 portal
protein are not required for phage viability (38, 39). Therefore, to
obtain sufficient material for in vitro studies, and to
simplify purification, a recombinant portal protein to which a
poly-histidine tag was appended to the C terminus was constructed and
overexpressed in E. coli. To verify that the His-tagged
protein was functional, the gene for the His-tagged protein was crossed
back into phage and the phage viability determined. Infectious phage
utilizing the His-tag were recovered (designated P22 1His). The burst
size of these phage was ~50% that of wild-type, indicating a slight
effect of the presence of the His-tag portal gene on phage
morphogenesis. The ability of phage 1His to plaque on P22-permissive
E. coli (40) was also verified.
Portal Protein Oligomerizes upon Storage--
Recombinant,
His-tagged portal protein was overexpressed in E. coli,
purified using nickel affinity chromatography, and analyzed by SDS-PAGE
(Fig. 1A). The expressed
protein was the dominant component of the lysate (Fig. 1A,
lane 2), was retained on the nickel column, and eluted as an
essentially pure protein at ~8 mg/ml (Fig. 1A, lane
5). A typical yield was 125 mg per 0.5 liter.
To determine the oligomerization state of the protein, an aliquot of
the protein was diluted to 1 mg/ml and injected onto an SW300 size
exclusion column (Fig. 1B). The sample eluted as two peaks:
an included peak comprising ~80% of the protein, an excluded peak
comprising ~10% of the protein. Species of intermediate sizes
comprised the remaining 10%.
To determine the effect of concentration on polymerization, one
aliquot of the purified protein was concentrated to 50 mg/ml, while
another was maintained at 1 mg/ml. After 18 h of incubation at
room temperature, the concentrated sample was diluted to 1 mg/ml, and
both samples were analyzed by SEC (Fig. 1B). The protein that had not been concentrated still contained primarily the smaller, included form. In contrast, the sample that had been concentrated to 50 mg/ml displayed a shift toward higher molecular weight species, suggesting the existence of a concentration-dependent
polymerization reaction.
The Small and Large Forms Can Be Isolated--
To isolate the
different forms of portal protein, the eluate from the nickel column
was dialyzed against low salt buffer (50 mM Tris-Cl, 50 mM NaCl, 20 mM 2-mercaptoethanol, 2 mM EDTA, pH 7.4), centrifuged to remove large aggregates,
loaded onto an anion exchange column, and eluted with an increasing
gradient of NaCl (Fig. 2A).
The protein resolved as a series of four dominant peaks eluting between
250 and 650 mM NaCl. Each peak was then examined by SEC
(Fig. 2B). The protein that had eluted at the lowest salt concentration (peak a: 250-320 mM)
eluted as a single peak that was fully included in the column matrix.
The protein that had eluted at the highest salt concentration
(peak d: 550-650 mM) eluted as a single peak
that was fully excluded from the column matrix. Protein that had eluted
at intermediate salt concentrations (peaks b and
c) contained a mixture of both the small and large forms.
In a separate experiment, the stability of the different forms was
determined. Samples from peaks a, b, and
d, were incubated at either 4 or 23 °C overnight at 0.2 mg/ml. Samples from peaks a and d were
unchanged, whereas the relative amount of small and large species in
the sample from peak b displayed a
temperature-dependent change in distribution. Low
temperature favored the smaller form. Presumably this shift in
distribution represented equilibration of long-lived intermediates to
the stable final forms.
The Two Stable Portal Protein Forms Are Monomers and
Rings--
Wild-type portal protein was previously isolated from
infected cells and characterized using SEC and rate-zonal sedimentation (8). From those data it was estimated that the unassembled form of the
protein was either a monomer or a dimer. Similarly, the unassembled
recombinant portal protein described here had an apparent molecular
mass of 175 kDa, which was estimated by comparison of its SEC elution
time to those of known standards (not shown). Although this was
consistent with this form having been a dimer of the 84-kDa portal
subunit, it was also possible that it represented an elongated monomer.
Therefore, to obtain the shape-independent molecular mass of the small
form of the protein, it was characterized using equilibrium analytical
ultracentrifugation in the same buffer used for the size exclusion
chromatography (Fig. 3A). The
best fit to a single species yielded a molecular mass of 87 kDa (± 4 kDa) with evenly distributed residuals. Attempts to fit the data with
alternate multimeric protein models were unsuccessful. SEC analysis of
the sample after the ultracentrifugation analysis verified that the
protein had remained unassembled during the experiment. Therefore, the
small form of the portal protein was an elongated monomer.
To determine the structure of the large form, a sample of protein that
had been eluted from the anion exchange column with 650 mM
NaCl was analyzed using negative-stain electron microscopy (Fig.
3B). The only visible structures were those of rings with outward-extending knobs. These structures were indistinguishable in
size and morphology from rings that formed from wild-type protein (8),
indicating that the large form of the protein was an oligomeric ring.
Portal Rings Assemble Slowly in Vitro--
To better characterize
the process of ring formation, the kinetics and thermodynamics of ring
assembly were determined. The observation that the rings were stable
once they had formed provided the opportunity to characterize their
formation from monomeric portal subunits in vitro.
The polymerization of monomers into rings was greatly retarded at low
temperatures; therefore, highly concentrated monomeric protein was
stored on ice. Polymerization was triggered by rapidly raising the
temperature by placing samples in a preheated block. Subsequently,
aliquots were removed, diluted to 0.1 mg/ml to prevent further
polymerization, and then analyzed using SEC (Fig.
4A). The amount of protein
assembled as rings at each time point was quantified and plotted (Fig.
4B). The resulting data points were then fit using a
single-exponential equation, and the apparent rate constant was
determined from the best fit to the data. At a concentration of 44 mg/ml, only about half of the subunits was converted to rings after
1.5 h at 25 °C. The rate constants were similarly determined at
15, 20, and 30 °C, and an Arrhenius plot was then generated (Fig.
4C). The slope of such a plot equals the negative value of
the activation energy. An activation energy of 12 kcal/mol of subunits
was obtained.
Portal Protein Changes Secondary Structure--
The conversion of
monomer to rings required a high concentration of protein and was slow
relative to the phage replication cycle (~45 min). This suggested
that the protein required a structural change to polymerize. To
determine whether there were changes in secondary structure when the
portal protein formed rings, the far-UV circular dichroism spectra of
purified monomer and rings were determined (Fig.
5). The CD spectra were collected at
25 °C at a concentration too low to promote ring formation during the measurements (0.2 mg/ml). The amount of monomer in the sample was
determined before and after the CD measurement and determined to be
~96% in both cases. The rings used for this experiment were intact
before and after the measurements as determined by SEC. The resultant
CD spectra indicated that there was a considerable increase in
secondary structure that accompanied ring formation. Both forms were
highly
The monomeric portal protein was estimated to contain 41% A New Domain Is Folded upon Ring Formation--
The increase in
The thermal transition was irreversible for both forms. In addition,
the CD spectra of the heated monomer and ring samples became super
imposable after these melting transitions occurred. The spectra of the
thermally denatured samples were different than either native spectrum.
Using the CDNN software, the heat-denatured samples were estimated to
contain ~31%
To determine whether aggregation indeed accompanied heat denaturation,
monomers and rings were heated to 30, 55, and 70 °C for 10 min and
the products were then analyzed by SEC (tandem TSK6000/SW300). Monomers
and rings both irreversibly aggregated above 55 °C, primarily to
filterable aggregates (>0.1 µm). Because the changes in secondary
structure that occurred upon heating the portal protein were
irreversible, we did not evaluate the melting curves thermodynamically.
The two cooperative transitions in the CD melting curve of the ring
supported the observation that the subunit had gained Bis-ANS Binds to the Monomer and Ring--
The in vitro
polymerization experiments revealed that the polymerization into rings
was greatly inhibited at low temperatures, suggesting a role for
hydrophobic interactions in the assembly of rings (42). However, both
monomers and rings were highly soluble at ambient temperatures,
suggesting that large hydrophobic regions were not surface-exposed. To
determine the nature of accessible hydrophobic regions of the monomer
and ring, each form was probed with the fluorescent dye bis-ANS. This
compound is a dimer of ANS and exhibits a very weak fluorescence in
polar solvents due to solvent quenching (35, 43). The fluorescence of
bis-ANS is greatly enhanced when the dye is bound within nonpolar
cavities and has been used extensively to monitor accessible
hydrophobic regions within proteins (37, 44-48).
Portal monomers and rings were incubated with increasing amounts of
bis-ANS. The fluorescence signal was corrected for background fluorescence as well as the inner-filter effect (35) and plotted to
represent the change in fluorescence as a function of bis-ANS concentration (Fig. 7). The observed
fluorescence is a function of both the binding constant and the
fluorescence enhancement accompanying binding (36, 49). At low
concentrations of bis-ANS, the observed fluorescence was higher for the
rings than for the monomers, whereas at high concentrations of bis-ANS
this pattern was reversed. Significantly, the binding of bis-ANS to the
rings approached saturation, whereas binding to the monomer did not (up
to 2.25 × 10
The nonsaturable binding behavior of the portal protein monomer is a
result of having many weak binding sites (49) and has been observed in
previous binding studies of bis-ANS and the chaperonin Cpn60 (50).
Furthermore, the binding to the monomers was nonhyperbolic, suggesting
that the binding of multiple bis-ANS molecules was altering the binding
capacity of the subunit (36), presumably by inducing a structural
change in the protein as has been reported for bis-ANS binding to other
proteins (46, 51). The inability to saturate the monomers with bis-ANS
prevented any determination of the total number of accessible binding
sites, because the fluorescence emission per bis-ANS binding event
could not be determined.
To minimize bis-ANS-induced structural perturbations, the binding to
the portal protein was characterized in the presence of excess protein
so that less than one bis-ANS molecule was bound per subunit (Fig. 7,
inset). Under these conditions binding only occurs at the
highest affinity sites (reviewed in Ref. 36). Fitting of these binding
data with a hyperbolic binding function revealed that the bis-ANS bound
the highest affinity site on the monomer with a Kd
of 0.7 µM, whereas the high affinity binding to the ring
had a Kd of 1.2 µM. In addition, the
ring titration of bis-ANS gave the same binding isotherm as the bis-ANS
titration of the rings, indicating that the rings bound only one
bis-ANS molecule per subunit. The highest affinity binding site on the
monomer had roughly twice the bis-ANS binding affinity of the rings.
However, the lower fluorescence enhancement (Fig. 7, inset,
lower Fmax) of bis-ANS bound to this site
suggested that it was more polar than the binding site on the rings.
This argues that a substantial hydrophobic surface was not
solvent-exposed on the monomer; yet, bis-ANS, when present at high
molar excess, entered the less polar, hydrophobic regions of the monomer.
Polymerization Stabilizes the Portal Protein--
The increase in
Fig. 8A shows the time course
of digestion of portal monomers cleaved by
The subunits in the rings were more resistant to chymotrypsin cleavage
than the monomers (Fig. 8B). After 5 min, the monomers had
all been cleaved to smaller fragments, whereas intact ring subunits
were detectable after 60 min. After 120 min of digestion, the ring
subunits had been cleaved to a ladder ranging from ~70 to 90 kDa. The
fragments C45 and N50 were also generated in
the digestion of the rings; however, fragment C45 was
generated more slowly. This suggests that the scissile bond connecting
fragments C45 and N50 was more protected in the rings.
In the presence of bis-ANS, the rate of digestion of the monomers and
rings was accelerated, indicating that bis-ANS was not inhibiting the
enzyme. In addition to the enhanced digestion of the intact subunit, no
detectable N-terminally derived fragments remained after 120 min of
digestion of the monomer. During the digestion of the rings in the
presence of bis-ANS, the cleavage that generated fragment
C45 was enhanced. This suggests that the binding of bis-ANS
was increasing accessibility to the scissile bond connecting
C45 and N50. In contrast to the digestion of
the monomer, N-terminally derived fragments were present after 120 min
of digestion in the presence of bis-ANS (fragment
N43, Fig. 8B). This presumably reflected a
structural change in this region that had occurred as a result of polymerization.
The digestion experiments revealed that the portal protein was more
resistant to proteolysis when assembled as rings, primarily at a
scissile bond in the subunit that connected two large N- and C-terminal
domains. Bis-ANS had apparently destabilized the N terminus of the
monomer upon binding. Once assembled as rings, this region was more
resistant to digestion in the presence of bis-ANS, suggesting that it
had become more stable. The fluorescence analysis of bis-ANS binding,
suggested that bis-ANS was denaturing a region of the monomer. Taken
together, these data imply that the monomer contained a meta-stable
domain that was stabilized upon ring formation.
Portal Protein Resists Polymerization--
During the expression
of the recombinant P22 portal protein, the intracellular concentration
was estimated to be greater than 100 times that observed during a
wild-type phage infection; however, the protein was recovered primarily
as a monomer. Because the rings were stable in the buffers used for
purification, we suspect that no dissociation occurred during
purification and that the protein was primarily unassembled in the cell
as well. The observation that portal protein rings did not readily
assemble in vivo is in agreement with the previous finding
by Bazinet and King (8) that unassembled P22 portal protein is isolated
from infected Salmonella defective for coat protein
production. The portal protein of bacteriophage SPP1, which has also
been over expressed in E. coli and characterized in
vitro, required divalent cations to form stable rings from
monomeric subunits, and so it was suggested that the intracellular
divalent cation concentration of the natural host (Bacillus
subtilis) was sufficient to drive ring assembly (52). The P22
portal protein was expressed in a host that can support phage
replication, suggesting that any requirements for ring assembly should
have been met during expression. The finding that P22 portal protein
rings did not appreciably form in vivo suggests that portal
ring formation is tightly coupled to phage head assembly.
Once purified, the portal protein assembled into rings, albeit more
slowly than required for phage morphogenesis. Kinetic measurements of
the polymerization of the portal rings indicated that appreciable ring
formation occurred only when the protein was highly concentrated. Even
at high concentrations, the protein took hours to approach equilibrium
implying that simple protein-protein collisions were not rate-limiting
(53, 54). Slow protein oligomerization processes are generally limited
by obligatory structural transformations that raise the activation
energy of association (55). The observation that the P22 portal protein
gained secondary structure upon polymerization suggests that this
structural rearrangement is responsible for at least part of the
observed 12 kcal/mol activation energy.
Protein Folding during Ring Formation--
There was a pronounced
structural change as the monomers became rings, primarily in the
formation of
The increase in secondary structure upon polymerization was accompanied
by the formation of a cooperatively folded domain that was not present
in the monomer. The folding of this domain may play a role in rendering
the assembly process irreversible as in the case of some protein
polymerization events (55, 58). It was determined that the monomer
contained a meta-stable domain near the N terminus that was perturbed
by bis-ANS binding. The monomers had the capacity to bind more bis-ANS
than the rings, suggesting that the bis-ANS had access to hydrophobic
regions within this domain that were inaccessible once the subunits had assembled. Indeed, some ring dissociation was observed after long incubations (>2 h) with excess bis-ANS, suggesting that bis-ANS was
binding in regions that formed inter-subunit contacts (data not shown).
An interesting aspect of the meta-stable domain in the monomer is that
it was resistant to proteolytic digestion, suggesting that it contained
a substantial amount of tertiary structure. This is supported by the
observation that the tryptophan fluorescence of the monomer is
blue-shifted relative to the ring, suggesting that this region is
solvent-protected.5 It is
tempting to speculate that stabilization of this meta-stable domain is
coupled to the observed folding increase.
A Meta-stable Monomer--
Meta-stable proteins have been
described as having little resistance to protease and are generally
referred to as being molten-globule-like because of their lack of
defined tertiary structure, similar to protein folding intermediates
(57). It has been postulated that this property allows some proteins to
adopt different conformations, because they bind to various regulatory
factors allowing a wide range of specificity and function (56, 57). In
addition, stabilizing the meta-stable regions in
Given that the P22 portal protein monomer is refractory to
polymerization in the absence of phage head assembly, it is conceivable that the subunit utilizes a meta-stable domain to refrain from premature ring formation. The observed activation barrier may be
coupled to leaving this intermediate conformation prior to committing
to the ring form. This mechanism could allow for strict temporal
control of assembly within the cell, because initiation of ring
assembly would require a "triggering" catalyst to lower the
activation barrier for assembly: akin to the process of
scaffold-catalyzed coat protein assembly observed in P22 (60) or the
initiation of TMV coat assembly by RNA (61). Once initiated,
autostery could then more efficiently recruit subunits to
assemble into the growing ring (62, 63).
Why Control Ring Assembly?--
Proteins destined to become
members of multisubunit assemblies can fold into inactive forms to
allow for kinetic control and pathway determination during assembly
(63-65). Additionally, kinetic control can be an integral requirement
for high fidelity assembly (66). Given that the P22 portal protein is
capable of assembling into rings by itself, the presence of a kinetic
barrier is intriguing. Perhaps preformed portal rings are detrimental
to viral morphogenesis. We have recently determined that preassembled
P22 portal rings are capable of stably binding to the P22 DNA packaging
machinery (gp2 and gp3).6 If
this binding represents aberrant DNA packaging initiation, then the
portal protein may be designed to prevent such catastrophic interactions by remaining unassembled until it is recruited into a
growing head.
The reduced assembly rate may also reflect an evolutionary solution to
an inherently difficult process: assembling the correct number of
subunits into the portal rings. The intrinsic curvature of a growing
ring may not be constrained sufficiently to ensure the production of
only one symmetry with a defined number of subunits (67) (assuming that
only one symmetry is utilized during viral morphogenesis). Indeed, it
has been observed that the portal protein of bacteriophage T7 can
assemble into rings with both 12- and 13-fold symmetry (68).
Additionally, the 13-subunit portal ring of bacteriophage SPP1
assembles through intermediates with a curvature that correlates with a
14-subunit ring (22). Efficiently assembling portal rings with a
defined number of subunits may require a scaffold to ensure the proper
ring diameter. Thus, some portal proteins may be designed such that
they only participate in assembly as members of an organizing complex.
In the case of the portal protein of bacteriophage T4 the product of
gene 40 serves this purpose (69). A model for portal vertex
formation during bacteriophage T7 morphogenesis has been proposed that
includes an interaction between the connector protein (p8) and the
outer shell protein (p10A) at an early stage of head assembly (70). In
this model, the connector assembles into a preformed hole in the capsid
lattice. Such a mechanism hints that the capsid hole could be a guide
for connector polymerization.
In the scheme of P22 head assembly, likely candidates for the role of
portal activator are the scaffold and coat proteins, because they
appear to be intimately involved in the assembly of the portal vertex.
It has been proposed that phage-encoded RNA may also play a role in
portal incorporation by serving as an organizing center (38). Mutants
in the P22 scaffolding protein have been isolated that are unable to
recruit portal protein into growing heads (71). Additionally, a mutant
portal protein (csH137), which is not recruited into
procapsids under restrictive conditions, can be rescued by several
different mutations in the P22 coat protein (26). This may reflect a
general mechanism for portal incorporation, because physical
interactions between scaffold, coat, and portal proteins have been
observed for other phages (72, 73).
-helicity upon
polymerization. Thermal denaturation studies suggested that the rings
contained an ordered domain that was not present in the unassembled
monomer. A combination of 4,4'-dianilino-1,1'-binapthyl-5,5'-disulfonic acid (bis-ANS) binding fluorescence studies and limited proteolysis revealed that the N-terminal portion of the unassembled subunit is
meta-stable and is susceptible to structural perturbation by bis-ANS.
In conjunction with previously obtained data on the behavior of the P22
portal protein, we propose an assembly model for P22 portal
rings that involves a meta-stable monomeric subunit.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
29, Lambda, T3, and SPP1
(18-22). Likewise, purified wild-type P22 portal protein spontaneously
formed rings with 12-fold rotational symmetry upon storage (8). Portal
rings represent a class of structures capable of fully surrounding DNA
in a nonspecific manner, analogous to the ring-like sliding clamps that
have been recognized as integral components of DNA replication
machinery (23, 24). A distinguishing feature of sliding clamps is that
they are topologically closed in their functional state and so, in the
absence of DNA threading, they must be assembled in place around the
DNA (23, 24). Little is known regarding the mechanism by which the
transformation from unassembled subunits to functional rings occurs. In
this manuscript we report a more detailed characterization of the
polymerization of P22 portal protein and provide insight into a
potential control mechanism for ring assembly.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
csH137
13
amH101 (26)) were grown on DB7000 containing
the recombination template at restrictive temperature. Plaques from the
template-rescued population were screened by immunoblotting with a
commercial antibody that recognized four consecutive histidines
(Qiagen, Valencia, CA). Phage that gave rise to positive immunoblots
were prepared as phage stocks, and the gene 1/gene
8 junction was sequenced to verify the presence of the
His-tag in gene 1.
-D-galactopyranoside, and shifted to
28 °C for 5-6 h. The cells were then harvested by centrifugation,
resuspended in 1/20 volume of cold nickel binding buffer (20 mM imidazole, 20 mM Tris-Cl, 500 mM
NaCl, pH 7.9) containing 0.2 mg/ml chicken egg white lysozyme, and
frozen at
70 °C. Induction levels were estimated by SDS-PAGE and
found to be ~300 mg of portal protein per liter of cells.
70 °C. Freezing and thawing the protein under these
conditions did not cause detectable aggregation nor did it cause
polymerization. The total yield of pure monomer was generally ~60 mg
per 500-ml culture determined spectrophotometrically in 6.0 M guanidine-HCl using a calculated extinction
coefficient of 99,740 M
1 cm
1
(28).
) using the equation y = y0 + Ae
x/
using
Origin 6.0 (Microcal). The logarithms of the rate constants (1/
)
from each fitted temperature series were plotted against reciprocal
temperature to generate an Arrhenius plot, and a straight line was
fitted to obtain the slope of the data. The activation energy was
obtained by calculating the negative of that slope by virtue of the
Arrhenius relationship: lnk =
Ea/RT + C (32), where
k is the rate constant, Ea is the
apparent activation energy, R is the gas constant, T is the
temperature, and C is a constant.
385 = 16,790 M
1 cm
1) (34). Fluorescence
measurements were made with a Shimadzu RF-1501 fluorometer (Shimadzu
Scientific, Columbia, MD) using
ex = 385 nm and
em = 485 nm. The fluorescence signal was corrected for
background bis-ANS fluorescence and correcting for the inner-filter effect (Fcorr = Fobs × 10(A385 + A485)/2]) (35). Fluorescence measurements of bis-ANS and protein were made after allowing the mixed samples to equilibrate for 30 min prior to measurement. The fluorescence signal was stable for solutions of
bis-ANS with monomers and rings.
-chymotrypsin (0.005 units/ml, Sigma
Chemical Co., St. Louis, MO) at room temperature in 50 mM
NaHPO4, 100 mM NaCl, pH 7.4. At defined time
points, aliquots were removed and placed into SDS sample buffer
containing 2 mM phenylmethylsulfonyl fluoride and
immediately heated to 100 °C for 5 min. These samples were then
resolved using SDS-PAGE using a 12.5% gel and stained with Coomassie Blue.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
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Fig. 1.
Purification of His-tagged portal protein
analyzed by SDS-PAGE. A, recombinant, His-tagged portal
protein was overexpressed in E. coli and purified using
nickel affinity chromatography. Lanes: procapsid,
purified P22 procapsids that contained the 84-kDa portal protein;
lysate, cleared bacterial lysate of induced expression host;
effluent, lysate material that did not bind to the nickel
column; wash, material removed with 65 mM
imidazole; eluate, material eluted with 500 mM
imidazole. Approximately 90% of the portal protein present in the
lysate was recovered in the elution step. B, samples of
purified portal protein were analyzed by SEC running in Tris buffer
(100 mM NaCl, 50 mM Tris-Cl, 2 mM
EDTA, pH 7.4). Samples: fresh eluate, a sample of
protein immediately after elution from the nickel column after dilution
in Tris buffer to 1.0 mg/ml; 1 mg/ml 18 h, protein that was
stored for 18 h at 1.0 mg/ml; 50 mg/ml 18 h, protein
that had been concentrated to 50 mg/ml for 18 h prior to dilution
to 1 mg/ml for SEC analysis.
View larger version (21K):
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Fig. 2.
Isolation of monomers and rings.
A, purified portal protein in 100 mM NaCl was
bound to an anion exchange column (Hi-Trap Q) and eluted with a NaCl
gradient from 200 to 700 mM NaCl in Tris buffer at pH 7.4. All of the protein bound to the column and was eluted between 250 and
650 mM NaCl. The dotted line is the total
gradient profile from 25 mM to 2 M NaCl.
B, selected fractions from the anion exchange column
characterized by SEC (SW300). Lowercase letters a
through d correlate fractions from the ion exchange column
with SEC chromatograms.
View larger version (62K):
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Fig. 3.
Assignment of the assembly state of the
portal protein. A, portal protein eluted with 320 mM NaCl from an anion exchange column was exchanged into
Tris buffer (100 mM NaCl, 50 mM Tris-Cl, 2 mM EDTA, pH 7.4) and allowed to reach equilibrium in an
analytical ultracentrifuge at three different concentrations at
4 °C. The equilibrium absorbance profiles were globally
analyzed to obtain a molecular weight estimation. Shown here is
the fit superimposed on representative absorbance data collected at 0.9 mg/ml. The residuals from this fit are plotted below.
B, negative-stain electron micrograph of the portal protein
eluted from the anion exchange column with 660 mM NaCl. The
sample was stained with 2% uranyl acetate. The scale bar
corresponds to 50 nm.
View larger version (27K):
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Fig. 4.
Ring assembly kinetics. A,
superimposed SEC chromatograms of portal protein at selected times
after the commencement of assembly at 25 °C at 520 µM
(44 mg/ml) in phosphate buffer. Solid lines are experimental
chromatograms, and dotted lines are chromatograms of
homogeneous monomer and ring used in the SVD-based quantification
procedure. B, a plot of portal protein assembled as rings at
various times at 25 °C. The data were fit using a single-exponential
equation (solid line) to obtain a macroscopic forward rate
constant. C, Arrhenius plot of rate constants from portal
polymerizing at 15, 20, 25, and 30 °C. A straight line
was fit to obtain the slope.
-helical as was evident by the spectral minima at 208 and 222 nm. To more accurately determine the secondary structural components
within each form, each spectrum was analyzed using the program
CDNN.
View larger version (22K):
[in a new window]
Fig. 5.
Far-UV CD spectra of the monomer and
ring. Averaged CD spectra of portal monomer (filled
circles) and rings (open circles) from 200 to 260 nm at
25 °C in phosphate buffer (100 mM NaCl, 30 mM NaHPO4, pH 7.4). The straight
lines are the smoothed data generated for calculating the amount
of secondary structure using the program CDNN. Monomer: 40.7%
-helix, 6.1% anti-parallel
-sheet, 4.8% parallel
-sheet,
18.7%
-turn, 27.9% random coil,
= 98.2%. Ring: 49.4%
-helix, 4.6% anti-parallel
-sheet, 4.9% parallel
-sheet,
15.9%
-turn, 25% random coil,
= 99.9%.
-helix,
whereas the ring contained 49%
-helix. The increase in
-helicity
was accompanied primarily by a loss in random coil. Computational
estimates of the absolute
-helical content of a protein are the most
accurate and are generally within ~5% of the amount determined from
high resolution structures (41). This error primarily arises from
errors in the determination of protein concentration (33, 41) and from
random noise (33). The CD spectra in Fig. 5 were averaged prior to
smoothing to reduce noise error. The concentrations of monomers and
rings, determined spectrophotometrically using denatured samples, were
within 1% of each other. Therefore, the calculated changes in
secondary structure upon ring formation are more accurate than the
absolute calculations of secondary structure, probably with less than
3% error.
-helix corresponded to roughly 6-7 kDa of peptide ordering or ~60
amino acids (~8% of the 83.6-kDa subunit). Because this ordering
involved a significant amount of the polypeptide backbone, it was
possible that a new cooperative folding unit had been generated upon
ring formation. To determine whether polymerization resulted in
cooperative stabilization of the
-helical secondary structure within
the subunit, both monomers and rings were thermally denatured, and the
-helical content of each was monitored by measuring the CD signal at
222 nm (Fig. 6). The monomeric protein exhibited a gradual loss of CD signal as the protein was heated from 2 to 37 °C. Between 37 and 45 °C, a single cooperative unfolding transition was observed that resulted in the loss of ~6% of the ellipticity at 222 nm. Once this transition had occurred, the CD signal
remained constant up to 98 °C. The rings exhibited a gradual loss of
CD signal from 2 to 43 °C. Two sharp melting transitions followed:
one from 43 to 51 °C, and another from 55 to 68 °C. When
combined, the amplitude of these two melting transitions accounted for
the difference in CD signal at 222 nm that existed between the monomer
and ring.
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Fig. 6.
Thermal denaturation of the monomer and
ring. Portal monomer (filled circles) and ring
(open circles) at 0.25 mg/ml were heated from 2 to 98 °C
in phosphate buffer, and the CD signal at 222 nm was recorded. Each
plot is the solvent-corrected average of two independently obtained
melting profiles using different preparations of monomers and
rings.
-helix at 25 °C. The loss of
-helicity from
the native forms was accompanied by an increase in antiparallel
-sheet (to 10%) and random coil (to 31%). These structural changes
were also accompanied by an increase in the turbidity of the samples
and suggested that the protein had formed large aggregates upon heating.
-helical
secondary structure upon ring formation; however, using circular
dichroism alone we could not determine which of the two cooperative
domains present in the rings was also present in the monomers.
Currently, we are characterizing the thermal and pressure stability of
both forms by monitoring tryptophan fluorescence in an attempt to
assign folded regions in the two forms.
5 M bis-ANS).
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Fig. 7.
Bis-ANS binding fluorescence. The
relative fluorescence of bis-ANS was recorded in the presence of 80 nM portal protein either as monomers (filled
circles) or assembled as rings (open circles) and
plotted ( F). Inset, the
fluorescence signal from 80 nM bis-ANS as it was titrated
with increasing amounts or either monomer (filled circles)
or rings (open circles). The solid lines
superimposed on these plots are the best fits of the hyperbolic binding
function that was used to obtain
Fmax.
-helicity, coupled with the alterations in melting behavior and
bis-ANS binding upon polymerization, suggested that ring assembly
resulted in stabilization of a domain of the portal protein. To
characterize stability changes that accompanied polymerization, both
monomers and rings were subjected to partial proteolysis in the
presence and absence of bis-ANS. The protein was digested at 23 °C,
and samples removed at time points ranging from 0 to 120 min were
treated with protease inhibitor and analyzed by SDS-PAGE. Fragments
containing the C terminus were identified by performing Western blots
using a monoclonal antibody that recognized the C-terminal His-tag
(bands labeled Cx, where x is
the approximate molecular mass in kilodaltons). To determine the origin
of bands that did not contain the His-tag, fragments were excised and
digested with trypsin, and the resultant peptides were identified by
mass spectroscopy.
-chymotrypsin. An initial
cleavage gave rise to two fragments of 45 and 50 kDa. These were
derived from the C- and N-terminal regions of the protein, respectively
(bands C45 and N50).
These two fragments were digested over the course of 120 min to yield
fragments C30 and N32. Tryptic peptides were
obtained from fragment N32 corresponding to amino acids in
the region spanning residues 15 to 145, thus placing the origin of
N32 at or very near the N terminus of the protein.
View larger version (103K):
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Fig. 8.
Proteolytic digestion in the presence of
bis-ANS. Portal protein (1.0 µM) was digested with
-chymotrypsin both with and without bis-ANS (100 µM)
present. A, monomer digestion fragments; B, ring
digestion fragments analyzed by SDS-PAGE. Lane labels
indicate digestion time in minutes and whether or not bis-ANS was
present. N and C denote fragments derived from
the N- and C-terminal halves of the protein, respectively.
Superscripts are approximate molecular masses in
kilodaltons. The black dots in B denote fragment
N43. Numbered tick marks on the sides
of each gel denote the positions and molecular masses in kilodaltons of
standards (Mark-12, Novex, San Diego) run on a separate gel with
selected digestion samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix. This is analogous to conformational changes
that can accompany protein-protein and protein DNA associations (56,
57). Recent characterization of the P22 portal protein using Raman
spectroscopy also revealed an increase in
-helical secondary
structure upon polymerization, although the effect appeared to be less
pronounced.3 Recently, a high
resolution structure of the
29 connector (portal) was determined by
x-ray crystallography and revealed substantial inter-subunit contacts
between
-helical regions.4
Although there is currently no high resolution structural information on the monomeric forms of any portal protein, it is not unreasonable to
assume that additional secondary structure forms as a result of
inter-subunit contacts.
-antitrypsin by
directed mutagenesis impeded protein function, suggesting that escape
from the meta-stable conformation can be coupled to biological activity
(59).
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ACKNOWLEDGEMENTS |
---|
We thank those who assisted in the preparation of these experiments: Sherwood Casjens (University of Utah, Salt Lake), Jonathan King (Massachusetts Institute of Technology), and Miriam Susskind (University of Southern California), for generously providing both bacterial and phage strains; Peter Weigele (University of Utah, Salt Lake) for assistance in designing and synthesizing some of the PCR primers; and Grace Wu (University of Alabama) for her technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health (NIH) Grant GM47980.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.
Supported by an NIH Basic Mechanisms in Virology pre-doctoral fellowship.
§ To whom correspondence should be addressed: Dept. of Microbiology, University of Alabama at Birmingham, BBRB Rm. 416, 845 South 19th St., Birmingham, AL 35205. Tel.: 205-975-5339; Fax: 205-975-5479; E-mail: prevelig@uab.edu.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M007702200
2 G. Bohm, personal communication.
3 G. Thomas, personal communication.
4 M. Rossman, personal communication.
5 S. D. Moore and P. E. Prevelige, Jr., unpublished observation. The P22 portal protein contains 11 tryptophans, 10 of which are located in the N-terminal half of the protein.
6 S. D. Moore and P. E. Prevelige, Jr., unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; SVD, singular value decomposition; CD, circular dichroism, bis-ANS, 4,4'-dianilino-1,1'-binapthyl-5,5'-disulfonic acid.
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REFERENCES |
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