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J Biol Chem, Vol. 274, Issue 23, 16135-16140, June 4, 1999
From the Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
The scaffolding proteins of double-stranded DNA
viruses are required for the polymerization of capsid subunits into
properly sized closed shells but are absent from the mature virions.
Phage P22 scaffolding subunits are elongated 33-kDa molecules that
copolymerize with coat subunits into icosahedral precursor shells and
subsequently exit from the precursor shell through channels in the
procapsid lattice to participate in further rounds of polymerization
and dissociation. Purified scaffolding subunits could be refolded in vitro after denaturation by high temperature or
guanidine hydrochloride solutions. The lack of coincidence of
fluorescence and circular dichroism signals indicated the presence of
at least one partially folded intermediate, suggesting that the protein
consisted of multiple domains. Proteolytic fragments containing the C
terminus were competent for copolymerization with capsid subunits into procapsid shells in vitro, whereas the N terminus was not
needed for this function. Proteolysis of partially denatured
scaffolding subunits indicated that it was the capsid-binding
C-terminal domain that unfolded at low temperatures and guanidinium
concentrations. The minimal stability of the coat-binding domain may
reflect its role in the conformational switching needed for icosahedral
shell assembly.
Scaffolding proteins are essential for the formation of the
icosahedral capsids of several classes of virus, including
double-stranded DNA bacteriophage (1-3), herpesviruses (4), and
adenoviruses (5). These proteins are required for assembly of the viral capsid but are not found in the mature virus after DNA packaging. In
the absence of scaffolding proteins, the viral coat protein polymerizes
into aberrant structures that cannot package DNA (1, 6-8).
In the presence of the 33-kDa scaffolding protein, the coat subunits of
the bacteriophage P22 form an icosahedral T = 7 lattice (9, 10),
which includes four distinct conformations of the coat monomers (8,
10). The scaffolding subunits are thought to be involved in the
conformational switching of coat subunits occurring during
polymerization at the edges of the growing shell (2, 3, 11).
Following successful procapsid assembly, on receipt of some
physiological signal (probably docking of the DNA packaging complex at
the portal vertex), all 200-300 scaffolding protein molecules dissociate from the procapsid lattice and recycle (12). The icosahedral
coat lattice exhibits 25 Å channels at the centers of the pentamers
and hexamers (9), which appear to be the site of exit for the
scaffolding subunits prior to DNA packaging. Soluble subunits can
reenter the procapsid through these channels in vitro (13).
Upon DNA packaging the capsid lattice expands, and the channels are
closed by a domain of the coat protein, which swings over to close the
channels (9, 14). In vitro the scaffolding subunits can be
released from procapsids by increasing temperature or by incubation
with very low levels of denaturant (13, 15).
This process may involve conformational changes in the scaffolding
protein as well as the coat protein. Within the assembled procapsid,
200-300 scaffolding protein molecules are relatively tightly packed
with each other into an inner shell or ball (10, 16). Scaffolding
subunits released from the procapsid, however, are highly soluble and
do not assemble any large structures in the absence of free coat
protein subunits (2, 17, 18). This presumably represents a
conformational change in the scaffolding subunits back to the precursor
conformation. Though direct structural evidence is limited, scaffolding
proteins appear to switch back and forth between alternate
conformations without phosphorylation, ATP, or other nucleotide binding steps.
Unfortunately, little is known about scaffolding protein structures.
Although the structures of both phage P22 and herpesvirus procapsids
containing scaffolding subunits have been determined, the scaffolding
organization was not revealed in either case, suggesting that it was
not icosahedral (10, 19).
Analysis of the sequences of both phage and herpesvirus scaffolding
proteins suggested that they tend to be predominantly Initial thermal denaturation studies of P22 procapsids by differential
scanning calorimetry revealed no evidence of a cooperative melting
transition for the scaffolding subunits (15). Preliminary nuclear
magnetic resonance studies of a 163-amino acid C-terminal fragment,
however, showed that stable elements of secondary structure were
present in solution (27). A recent study of scaffolding protein thermal
denaturation followed by Raman spectroscopy revealed that the protein
lost its helical secondary structure over a broad temperature range in
a noncooperative manner and appeared to have little packed tertiary
structure (28). The authors proposed that the scaffolding protein
consisted of several loosely packed helical segments.
These segments might correspond to functional domains for the various
roles of scaffolding protein. In addition to morphogenesis, the process
by which the double-stranded DNA phages ensure the incorporation of a
DNA packaging portal at a unique vertex also requires scaffolding
protein. Interactions between scaffolding and portal proteins have been
inferred genetically (29-31) or observed directly, in the case of
We were particularly interested in determining the scaffolding protein
region involved in binding to the coat protein. Also of interest was
the possibility that this cycle of association and dissociation was
coupled to the folding and unfolding of domains of the scaffolding
subunits. Accordingly, we have purified and characterized the
denaturation transition of soluble wild type scaffolding subunits. In
the accompanying paper (36) we describe a set of mutant scaffolding
proteins which are defective in aspects of shell assembly or function.
Chemicals--
Ultrapure
GuHCl1 was purchased from
Pierce. Proteases (V8, chymotrypsin-agarose, trypsin-agarose) and
inhibitors were ordered from Sigma. Molecular weight standards were
purchased from Bio-Rad. All other chemicals were reagent grade from
common sources.
P22 Phage Strains--
The phage strains used in the preparation
of wild type proteins carried the c1-7 mutation for lytic
growth and two nonsense mutations, 2amH202, in one of the
DNA packaging genes to block DNA packaging, and 13amH101,
which delays lysis.
Purification of Scaffolding and Coat Proteins--
Wild type
procapsids were purified from cells infected with a phage mutant
blocked in DNA packaging, and both scaffolding monomers and empty coat
shells were purified as described previously (2). The scaffolding
protein was dialyzed into phosphate buffer (20 mM
K2HPO4, 25 mM NaCl, pH 7.6, with
HCl) before use. The concentration was checked by absorbance at 280 nm,
based on an extinction coefficient of 1.61 × 104
liter mol Thermal Denaturation--
Wild type scaffolding protein was
diluted to 100 µg/ml (0.3 µM) in phosphate/NaCl buffer
before use. Fresh samples were prepared for each experiment.
Fluoresence measurements were performed using a Hitachi F4500
spectrofluorimeter interfaced with a personal computer. The excitation
wavelength was set to 280 nm, and the emission wavelength was 330 nm.
The excitation and emission slit widths were set to 2.5 and 5.0 nm, and
the PMT voltage was set to 700 V. Circular dichroism measurements were
made using an Aviv model 60DS spectropolarimeter with a
computer-controlled Peltier thermostat. Circular dichroism ellipticity
at 222 nm was measured for samples in a 0.5-cm-pathlength cell. The
spectral bandwidth was 1.5 nm, and the averaging time was 30 s.
For the fluorescence experiments the temperature of the controlling
water bath was adjusted in 2 °C steps from 3 to 89 °C, and the
sample was equilibrated at each temperature until the reading was
constant before recording the fluorescence intensity. For the circular
dichroism experiments, the temperature was automatically adjusted in
1 °C steps from 3 to 90 °C with 2 min of equilibration at each
point after reaching constant temperature. At the end of each
experiment the sample was cooled back to 3 °C to check for recovery
of the original signal.
The fraction folded was determined by the formula
(Xobs GuHCl-induced Denaturation--
Wild type scaffolding protein
was added to tubes containing varying concentrations of GuHCl in the
phosphate/NaCl buffer to give a final protein concentration of 100 µg/ml (0.3 µM) and stored for at least 12 h at
4 °C. The signal from these samples remained constant up to 24 h later, demonstrating that they had reached equilibrium. The same
samples were assayed by both fluorescence and CD and compared with
blanks of the same GuHCl concentration. Denaturation was monitored by
either fluorescence intensity at 330 nm with excitation at 280 nm or by
circular dichroism ellipticity at 222 nm as described above for the
thermal denaturation. Because even low concentrations of GuHCl caused
unfolding of scaffolding protein as measured by CD, the CD of
scaffolding protein in the absence of GuHCl was used as
Xn for calculation of the CD denaturation curve.
The temperature of the cuvette was maintained at 10 °C.
Proteolysis--
Wild type scaffolding protein was dialyzed into
20 mM phosphate buffer, pH 7.6, with phosphoric acid. The
scaffolding protein solution was diluted to a final concentration of
0.6 mg/ml (1.8 µM) in either phosphate buffer or
phosphate buffer with GuHCl and incubated at 10 °C for 2 h to
allow unfolding to occur. 2.5 µg of protease V8 was added to give a
1:10 ratio of protease to scaffolding protein. The samples were mixed
on a Nutator platform rocker for the designated amounts of time.
Proteolysis was halted by addition of trichloroacetic acid to 10%. The
precipitated samples were pelleted by a 4-min spin in an Eppendorf
microcentrifuge, and the pellets washed with acetone to remove the
acid. Samples were analyzed by SDS-polyacrylamide gel electrophoresis
on a 10% acrylamide gel.
Assembly and Binding of Proteolytic Fragments--
Wild type
scaffolding protein was diluted to 1 mg/ml (3 µM) in 200 µl of phosphate buffer. 20 µl of either trypsin or chymotrypsin conjugated to agarose beads as a 1:1 slurry in buffer was added, and
the tubes were mixed on a Nutator platform rocker. After 6 min, the
trypsin sample was centrifuged to pellet the beads, and the supernatant
was removed to a tube containing 3 µl of 1 mg/ml bovine pancreatic
trypsin inhibitor. The chymotrypsin sample was centrifuged after 10 min, and the supernatant added to 3 µl of 20 mg/ml
N-tosyl-L-phenylalanine chloromethyl ketone. To
these digests were added either empty procapsid shells, for a final concentration of 1 mg/ml (53 nM), or coat monomers to 0.5 mg/ml (1.1 µM). These reactions were incubated at room
temperature for 2 h. The samples were then centrifuged through
5-20% sucrose gradients for 35 min at 35 K. 18 fractions were
collected from each gradient through a pinhole at the bottom of the
tube. The protein compositions of the fractions were determined by
SDS-polyacrylamide gel electrophoresis on 15% acrylamide gels.
N-terminal Sequencing--
Scaffolding protein fragments were
generated with protease V8, trypsin, or chymotrypsin as described and
separated by SDS-polyacrylamide gel electrophoresis. The peptide
fragments were transferred to polyvinylidine difluoride membranes
(Immobilon polyvinylidene difluoride, Millipore) according to the
method of Matsudaira (39). The transfer was performed in a Hoefer
transfer unit at 100 mA for 16 h at room temperature. The membrane
was stained by Coomassie Blue to reveal the location of the transferred
peptide fragments. The 7 N-terminal residues of each selected band were
sequenced by the MIT Biopolymers Laboratory using an Applied Biosystems model 477A Perkin-Elmer Sequencer with an on-line model 120 PTH Amino
Acid Analyzer.
Thermal Denaturation of Wild type Scaffolding Protein--
To
detect possible folding domains of the scaffolding protein, the thermal
denaturation of purified scaffolding protein was monitored using two
signals: fluorescence at 330 nm, which measures the solvent exposure of
the single tryptophan residue, amino acid 134, and circular dichroism
at 222 nm, a signal of
The thermal denaturation curve observed by CD did not coincide with
that monitored by the fluorescence signal (Fig.
2), demonstrating the presence of at
least one partially folded intermediate. The CD data were similar to
that observed by Tuma et al. (28) despite the fact that the
concentration of scaffolding used in their experiment was 18-fold
greater than that used here. In that case, the authors fit the data as
a single two-state transition. Although our CD data were also well fit
as a single transition with a Tm of 46 °C,
the noncoincidence of the fluorescence curve (with a
Tm of 58 °C) reveals that there must actually
be multiple steps in the denaturation process.
A portion of the scaffolding protein began to lose secondary structure
at 15 °C. Approximately one-third of the scaffolding protein
secondary structure is GuHCl-induced Reversible Denaturation of Wild Type Scaffolding
Protein--
Low concentrations of GuHCl are sufficient to extract
scaffolding protein from procapsids (13, 17). To determine whether GuHCl causes extraction by denaturing that part of the scaffolding protein required for binding to the coat shell, we investigated the
effects of GuHCl on the stability of purified scaffolding protein.
Scaffolding protein was unfolded by incubation in concentrations of
GuHCl from 0 to 4 M for at least 12 h, after which
time the samples had reached equilibrium and no further spectral
changes occurred. The experiments were carried out at 10 °C to
remain below the temperature at which thermal unfolding begins.
The unfolding curves measured by CD and by fluorescence did not
coincide, demonstrating once again that the unfolding process was not
two-state and consistent with the presence of more than one domain. As
shown in Fig. 3, the loss of secondary
structure began upon the addition of minimal amounts of denaturant. The fluorescence transition did not begin until GuHCl concentrations at
which about 40% of the helical secondary structure had already been
lost. As the fluorescence signal is due to a single tryptophan at
residue 134, one explanation is that the fluorescence signal monitors
the unfolding of a particular folding domain containing the tryptophan
that is more stable than other regions of the protein. Because a GuHCl
concentration of only 0.5 M is sufficient to extract all
the scaffolding from the coat shells (13), this could be the domain
required for mediating binding of scaffolding to the coat shell. This
domain presumably does not include the region surrounding tryptophan
134.
Dilution of the protein out of GuHCl led to complete recovery of the
original signal, demonstrating that the GuHCl-induced unfolding was
reversible. The efficient refolding of the scaffolding protein without
interference from competing aggregation reactions was quite distinct
from the in vitro refolding of the coat protein and the
tailspike proteins. For both of these proteins, off pathway association
reactions competed with productive refolding (40-44).
Proteolytic Digestion of Folded and Partially Unfolded Scaffolding
Protein--
The lack of coincidence between the CD and fluorescence
signals indicated the presence of a partially folded intermediate during both thermal and GuHCl-induced denaturation transitions. This
suggests that the native state may consist of distinct
folding/unfolding domains. We were particularly interested in the
region of the molecule that unfolds at low GuHCl concentrations,
because this might be a region required for binding to the coat shell.
To determine which part of the scaffolding protein unfolded first, we
looked for regions of the molecule that became more accessible to
protease V8 after incubation in low concentrations of GuHCl at
10 °C. Protease V8 was chosen because it is resistant to
denaturation by GuHCl (45).
The protease digestion patterns of native and partially unfolded
scaffolding protein are shown in Fig. 4.
Under the buffer conditions used, protease V8 cleaves after both
glutamic and aspartic acid residues. As the amount of GuHCl increased,
the pattern of cleavage changed, with new bands becoming more
prominent. The action of the protease was also inhibited, requiring
longer digestion times, but this inhibition was due to the chloride
ions, which inhibit V8, not the guanidinium. As shown in the two
far right lanes of Fig. 4, the presence of 1.5 M NaCl
had the same inhibitory effect as the GuHCl, but the pattern of
cleavages was not altered, demonstrating that the exposure of new
cleavage sites was indeed due to denaturation of the scaffolding
protein by GuHCl.
The major cleavage bands for both folded and partially unfolded
scaffolding protein were blotted onto polyvinylidene difluoride membranes, and their N termini were sequenced to determine the location
of the cleavage sites. The size of each fragment was calculated based
on a calibration with a set of molecular weight standards, so that in
cases where the cleavage was in the C terminus, the cleavage site could
be estimated by comparing the size of the fragment to that of the
intact protein. The fragments that were sequenced, and their determined
or estimated cleavage sites are shown in Table
I. The three new sites exposed by GuHCl
denaturation (Glu221, Asp261, and
Asp267) were all in the C-terminal half of the molecule,
indicating that a C-terminal region unfolded first. This result was
consistent with the location of the tryptophan residue in the
N-terminal half of the protein.
Function of Proteolytic Fragments--
To test the assignment of
the C terminus as the essential end for binding, we prepared
proteolytic scaffolding protein fragments. These were tested for their
ability to copolymerize with the coat protein into procapsid-like
shells. Fragments were generated with either trypsin or chymotrypsin
rather than V8, because inhibitors were available to stop the
proteolysis without denaturing the scaffolding. Samples of scaffolding
protein were digested for a given amount of time with each protease
before addition of the appropriate inhibitor to stop the proteolysis.
Two assays were carried out to test the ability of the fragments to
interact with coat subunits. In the first, the entire digest was mixed
with purified empty procapsid shells to test whether the fragments
could enter and stably bind to the procapsid lattice. Complete
scaffolding protein molecules efficiently re-enter empty procapsids and
refill the interior space (13). In the second assay the fragments were
incubated with monomeric coat subunits to test for assembly of
procapsids in vitro. The reactions were allowed to proceed
for 2 h and then centrifuged through sucrose gradients to separate
scaffolding fragments stably associated with procapsids from those
remaining as monomers.
Digestion of scaffolding protein by chymotrypsin resulted in only one
cleavage, generating a large fragment of 30 kDa and a small one of 10 kDa. The large chymotryptic fragment was active in the reentry assay
and was recovered associated with shell structures in the middle
fractions of the sucrose gradient shown in Fig. 5b. The large fragment also
copolymerized with coat subunits, forming procapsid shells in
vitro, as shown in Fig.
6b. The small fragment did not
associate with the capsids under any conditions and remained at the top
of the sucrose gradients.
Trypsin digestion of scaffolding protein resulted in a range of bands,
from approximately 35 to 10 kDa. The bands from the trypsin digest
migrating at 30 kDa were able to associate with the coat shells in both
assays, whereas a larger band migrating at 35 kDa was not active in
either assay (Figs. 5a and 6a).
The 30-kDa chymotrypsin band, and the 30- and 35-kDa trypsin bands were
chosen for N-terminal sequencing. Both the 30-kDa bands had lost their
N termini, from cleavages at Tyr63 and Arg65.
The 35-kDa fragment had an intact N terminus and was estimated to have
lost approximately 20-30 residues from the C terminus. These results
demonstrated that the scaffolding C terminus was essential for any
interaction with the coat protein, whereas an N-terminal region of at
least 65 residues was dispensable, at least in the presence of
full-length scaffolding.
Scaffolding Protein Contains Multiple Domains--
The unfolding
of P22 scaffolding protein to an apparent equilibrium by both GuHCl and
heat revealed a transition that was definitely not two-state. Two
signals, fluorescence and CD, failed to give coincident transitions. In
both cases, the transition monitored by fluorescence lagged behind that
monitored by CD. Because the CD signal is due to secondary structure,
in this case
This analysis is supported by the results reported in the accompanying
paper for the denaturation of the Y214W mutant protein, which has an
additional tryptophan (36). This protein displayed an extra, lower
temperature transition by fluorescence.
Identification of the Coat-binding Region--
Because low
concentrations of GuHCl are sufficient to disrupt the binding
interactions between scaffolding protein and coat shells, it is
reasonable to conclude that the part of the molecule unfolded at these
GuHCl concentrations is the region involved in binding. Identification
of a region within the C-terminal half of the protein as the binding
region was confirmed by the behavior of proteolytic scaffolding protein
fragments in the shell binding and assembly assays; loss of the
C-terminal 20-30 residues made the scaffolding inactive, whereas loss
of the 65 N-terminal residues had no effect.
Parker et al. (47) have recently reported the results of
similar experiments using scaffolding protein fragments expressed from
cloned genes. A scaffolding fragment lacking 140 residues from the N
terminus was still active in assembly assays, whereas a fragment
lacking the C-terminal 10 amino acids was inactive. These results
together with those reported here establish that the C-terminal domain
of the scaffolding subunit is necessary and sufficient to direct the
assembly of the coat subunits in vitro.
Similar results have been obtained for other viral scaffolding
proteins. A long amber mutant of the major scaffolding protein of
bacteriophage T4, gp22, was able to assemble into naked cores in
vivo. Although wild type coat protein was present in these infections, it did not cover these cores but formed polyheads as if
scaffolding protein were absent (48). The authors suggested that the T4
scaffolding protein consisted of two domains, of which the C-terminal
domain was required for attachment of the core to the coat shell.
Mutations within the C-terminal 10 amino acids of the
In the case of herpesvirus scaffolding proteins, cleavage of a
C-terminal 25-amino acid segment is required for scaffolding release
and DNA packaging (50). Scaffolding proteins lacking this region
produced aberrant procapsids when expressed in cells, resembling those
produced in the absence of scaffolding protein (6, 7). Studies of
herpesvirus scaffolding proteins in vitro have demonstrated
that residues within the C-terminal 25 amino acids are both necessary
and sufficient for binding to coat protein (51-53). A cyclic analogue
of the C-terminal peptide bound coat protein much more efficiently than
a linear version (51, 52), suggesting that helicity of this region is
required for binding, consistent with the results presented here for
P22. An essential coat-binding helical switch segment at the
scaffolding protein C terminus may be a conserved feature among both
phage and animal viruses.
The N terminus of the P22 scaffolding protein is involved in
post-transcriptional repression of scaffolding synthesis (33, 34, 53),
probably by binding to the mRNA near the site of its translational
initiation. Based on the behavior of scaffolding amber fragments, the
region up to residue 78 was determined to be sufficient for
translational repression (54). A model has been proposed in which a
complex of scaffolding molecules bound to mRNA serves as the
initiator of procapsid assembly (55). All three proteases used in these
experiments cleaved the scaffolding protein between residues 60 and 65. This segment may be an exposed loop marking the end of an
mRNA-binding domain consisting of the 60 N-terminal residues.
Why Is the Scaffolding Protein So Unstable?--
The P22
scaffolding protein is an unusually unstable protein that begins to
lose secondary structure upon addition of the smallest amount of
denaturant and at surprisingly low temperatures. The coat protein of
P22 is equally unstable to denaturant (42). It may be that unassembled
viral proteins must be conformationally flexible to permit
conformational changes required during the assembly process. The
crystal structures of the RNA plant viruses (56) as well as SV40 showed
that the viral capsomeres are held together by entwined arms (57).
These arms could not have been folded before assembly, because they
would not have been free to make such extensive interactions. A similar
flexible arm might attach the scaffolding protein to the coat protein
shell. Consistent with this analysis, the C-terminal regions of the
scaffolding protein, which contain the coat-binding domain, are the
least stable.
In addition to low stability of the secondary structure, the
scaffolding protein appears to have little packed tertiary structure, because all peptide amide groups are available to rapid deuterium exchange (28). Consistent with a large exposure of nonpolar residues,
each scaffolding monomer binds 12-16 molecules of
1,1'-bi(4-anilino)naphthalene-5-sulfonic acid (bisANS) (23), a small
dye used to probe the accessibility of hydrophobic surfaces on proteins
(58).
Although these features are characteristic of a molten globule (59),
the scaffolding protein does display what appears to be cooperative
unfolding of distinct regions. The scaffolding protein simply may not
have a hydrophobic core as for typical globular proteins. Perhaps it
consists of a series of loosely interacting helices. Tight interactions
of exposed helical surfaces are probably formed within the closely
packed interior of the procapsid. The formation of these interactions
might be a driving force in procapsid assembly. In this sense, the
final product of the scaffolding protein folding pathway is not an
individual scaffolding subunit but an assembled procapsid.
It is interesting that the microtubule-binding protein tau is highly
extended and contains almost no secondary structure in solution (60),
leading to the suggestion that it exists in a "natively denatured"
state (61). The MAP-2 protein has similar properties (62). Although
scaffolding protein does have substantial secondary structure, the
proteins are similar in that all can function despite lacking the
characteristics of folded proteins. The scaffolding protein may
represent a class of proteins that are not meant to lead an independent
existence and thus do not require the same degree of structure as a
typical soluble enzyme.
*
This work was supported by National Institutes of Health
Grant GM17.890, a National Institutes of Health Biotechnology Training Grant, and a fellowship from the W. M. Keck Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviation used is:
GuHCl, guanidine
hydrochloride.
In Vitro Unfolding/Refolding of Wild Type Phage P22
Scaffolding Protein Reveals Capsid-binding Domain*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical in
structure (20, 21). This prediction has been confirmed for the P22
scaffolding protein by Raman spectroscopy (23) and circular dichroism
(24). Because there are no leucine zipper or heptad repeats evident in
the P22 scaffolding protein sequence (22), the protein is probably not
a coiled-coil. Analytical ultracentrifugation or gel filtration have
revealed that the scaffolding proteins of T4, 
, and P22 are all
highly elongated molecules (18, 24-26). Parker et al. (26)
estimated dimensions for the P22 scaffolding protein of 247 Å in
length by 22 Å in diameter.
29 (32). The unassembled P22 scaffolding protein regulates its own
synthesis at the translational level (33, 34) presumably by binding to
its mRNA. Mutations within a specific region of the P22 scaffolding
protein affect the ability of the scaffolding protein to be released
from the procapsid (35), suggesting that release or sensing of DNA
entry may be another scaffolding protein function. Fig.
1 summarizes the roles of the scaffolding
protein in procapsid assembly and DNA packaging that have been
identified in previous studies.
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Fig. 1.
Functions of scaffolding protein in the
assembly pathway of bacteriophage P22. Mutant substitutions in the
scaffolding protein are listed underneath the assembly steps they
affect.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 cm1 (23). Shells of coat protein
were dissociated into monomers by incubation in 2.0 M GuHCl
and loaded onto a Biogel A0.5 column to separate the monomers from any
remaining intact shells. The coat monomers were dialyzed overnight at a
concentration of approximately 1.0 mg/ml to remove the guanidine. The
concentration of the coat monomers was determined by measuring
absorbance at 280 nm, based on an extinction coefficient of 4.45 × 104 liter mol1 cm1 (23).
Xu)/(Xn Xu), where Xn is the
fluorescence or CD of the native protein, Xobs
is the observed value, and Xu is the fully
denatured value. Xn and
Xu were determined by linear extrapolation of
the folded and unfolded base lines into the region of the transition at
each temperature (37). This procedure is required to correct for the
intrinsic temperature dependence of tryptophan fluorescence, which in
these experiments accounted for 16% of the total signal change. The
slopes of the base lines were determined by linear least squares
analysis using Kaleidagraph software (Abelbeck). The corrected data
were fit to an equation for either a two-state transition (38) or the
sum of two or three simultaneous two-state transitions. To limit the
number of variables in these equations, separate folded and unfolded
base lines for each of the two or three transitions were not determined
but were assumed to be accounted for by the initial correction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical structure. The concentration of
scaffolding protein was 100 µg/ml (0.3 µM), a
concentration at which the protein should be almost entirely monomeric
(26).
View larger version (13K):
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Fig. 2.
Thermal denaturation of wild type scaffolding
protein. Wild type scaffolding protein at 100 µg/ml (0.3 µM) was heated from 3 to 90 °C, and the unfolding
process was monitored by fluorescence emission at 330 nm ( 
) with the
excitation at 280 or by circular dichroism at 222 nm (
). The
percentage folded was calculated as described under "Experimental
Procedures." The solid lines represent fits of the data to
a two-state transition model.
-helical, the rest being predominantly coil
and turns (28, 36). About 13% of this -helical structure had been
lost by 30 °C, the normal temperature of growth in
vivo.
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Fig. 3.
GuHCl-induced unfolding of wild type
scaffolding protein. Wild type scaffolding protein was diluted to
100 µg/ml (0.3 µM) in varied concentrations of buffered
GuHCl. When equilibrium was reached after incubation overnight, the
percentage folded was determined as described under "Experimental
Procedures" from the fluorescence emission at 330 nm ( ) when
excited at 280 nm and the circular dichroism ellipticity at 222 nm
(). Both curves include data points from three
independent experiments. The solid lines represent fits of
the data to a two-state transition model.
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Fig. 4.
V8 proteolysis of native or partially
denatured scaffolding protein. Scaffolding protein at 0.6 mg/ml
was incubated with protease V8 at 10 °C in buffer or buffer with
GuHCl (or NaCl) added as described under "Experimental Procedures."
After the times indicated the reactions were stopped, and the samples
were analyzed by SDS-polyacrylamide gel electrophoresis.
Protease V8 cleavage sites in the presence and absence of GuHCl
View larger version (90K):
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Fig. 5.
Binding of scaffolding protein fragments to
empty procapsid shells. Scaffolding protein at 1 mg/ml (3 µM) was digested by either trypsin (a) or
chymotrypsin (b) as described under "Experimental
Procedures." Empty procapsid shells were added to 200 µl of each
digest for a final concentration of 1 mg/ml (53 nM). The
reactions were incubated at room temperature for 2 h and then
centrifuged through 5-20% sucrose gradients for 35 min at 35 K. 18 fractions were collected from each gradient through a pinhole at the
bottom of the tube. The fractions were run on 15% SDS gels, and the
gels were stained by Coomassie Blue.
View larger version (94K):
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Fig. 6.
In vitro assembly of scaffolding
protein fragments into procapsids. Scaffolding protein at 1 mg/ml
(3 µM) that had been digested by either trypsin
(a) or chymotrypsin (b) as described under
"Experimental Procedures." Coat monomers were added to 200 µl of
each digest to give final coat protein concentration of 0.5 mg/ml (10.5 µM). The reactions were incubated at room temperature for
2 h and then centrifuged through 5-20% sucrose gradients for 35 min at 35 K. 18 fractions were collected from each gradient through a
pinhole at the bottom of the tube. The fractions were run on 15% SDS
gels. The gels were stained by Coomassie Blue.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix, whereas the fluorescence signal indicates the
local environment of the single tryptophan residue, the fluorescence
signal would be expected to drop first if scaffolding protein
denaturation proceeded through a molten globule intermediate (46). That
some secondary structure was lost before any change in fluorescence was
detected implies that the intermediates retained some tightly packed
structure. These intermediates are therefore not molten globules but
molecules in which only particular domains have unfolded.
29 scaffolding
protein removed its ability to interact with the coat protein when
expressed in vivo (49).
FOOTNOTES
Present address: G. W. Hooper Foundation, UCSF, San
Francisco, CA 94143.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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