From the
Temperature-sensitive folding (tsf) mutants of the phage P22
coat protein prevent newly synthesized polypeptide chains from reaching
the conformation competent for capsid assembly in cells, and can be
rescued by the GroEL chaperone (Gordon, C., Sather, S., Casjens, S.,
and King, J.(1994) J. Biol. Chem. 269, 27941-27951).
Here we investigate the stabilities of wild-type and four tsf mutant
unpolymerized subunits. Wild-type coat protein subunits denatured at 40
°C, with a calorimetric enthalpy of approximately 600 kJ/mol.
Comparison with coat protein denaturation within the shell lattice (T
Viral coat protein molecules in their mature conformations
interact intimately, forming a shell that encloses the viral nucleic
acid. For chemically identical subunits, this often requires that they
assume several distinct conformations within the shell (Caspar and
Klug, 1962; Harrison, 1984; Liddington et al., 1991). Little
is known of the conformation of viral capsid subunits before they
polymerize into shells.
Double-stranded DNA bacteriophages as well
as some eukaryotic viruses are not formed by a simple condensation
reaction of the coat protein with nucleic acid (Hendrix, 1985; Newcomb
and Brown, 1991; Horwitz, 1991). Instead, a precursor shell, the
procapsid, is first formed (Fig. 1). Procapsid particles contain
a shell of coat protein surrounding a core of virally encoded
scaffolding protein. The scaffolding protein molecules exit the
procapsid as DNA is pumped in through a unique vertex, and the viral
shell expands (Earnshaw and Casjens, 1980; Prasad et al.,
1993).
Studies by x-ray scattering and more recently by cryo-electron
microscopy have demonstrated that the P22 procapsid is smaller and
rounder than the virion particle (Earnshaw et al., 1976;
Prasad et al., 1993). The coat protein is arranged in an
icosahedral T = 7 lattice of pentamers and hexamers on
the procapsid shell and in the mature phage. This suggests that the
coat protein can exist in 14 different conformations while on the shell
(Caspar and Klug, 1962; Prasad et al., 1993). Holes present at
the center of procapsid hexamers and pentamers are thought to represent
the exit port of the scaffolding protein (Greene and King, 1994).
Rearrangement of coat protein mass seals these holes in the phage
(Prasad et al., 1993).
For many oligomeric proteins, chain
folding is linked to subunit assembly, which occurs from partially
folded intermediates (Jaenicke, 1987). It is unclear whether virus
capsids assemble from partially folded molecules that complete folding
during the assembly process, or fold into their lattice conformations
in solution prior to subunit association.
The coat protein can be
refolded in vitro from the fully denatured state, and the resultant
subunits are monomeric and competent for assembly in vitro (Teschke and
King, 1993). As a means of investigating the coat protein folding and
assembly process, we studied a set of 18 coat protein
temperature-sensitive mutants in vivo (Gordon and King, 1993).
These missense mutations, resulting in single amino acid substitutions,
were distributed throughout the gene and had in vivo restrictive growth temperatures ranging from about 33 °C to 41
°C (Gordon and King, 1994; Gordon et al., 1994). All of
the mutants had similar phenotypes. Virion particles formed at low
temperature were stable at restrictive temperature. When cells were
infected at restrictive temperature, the mutant coat protein was
recovered in inclusion body aggregates. The formation of irreversible
aggregates generally reflects poor stability of folding intermediates
rather than of the native folded molecule (London et al.,
1974; Mitraki and King, 1989; Goldberg et al., 1991). These
results, as well as additional genetic studies (Gordon and King, 1994),
suggested that the mutants were defective in folding.
The
temperature-sensitive folding phenotype of these mutants was corrected
by increasing the levels of GroEL/S, which transiently interacted with
newly synthesized coat protein (Gordon et al., 1994). These
data suggested that thermolabile intermediates were physiological
substrates for GroEL/S in vivo. The T4 major coat protein also
requires GroEL function, and in fact the phage T4 encodes a specific
homolog of GroES (van der Vies et al., 1994).
The
stabilities of P22 coat subunits and the procapsid shell have also been
studied using hydrostatic pressure (Prevelige et al., 1994).
Two single-site substitutions, including W48Q, had marked effects on
the stability of the assembled shell (Foguel et al., 1995).
Here we report characterization of purified protein subunits from
wild-type and four of the tsf
Empty procapsids were purified from procapsids as described by
Galisteo and King(1993), with minor modifications. Procapsids in the
presence of 0.5 M GdmCl were run through a Bio-Gel A-0.5m
column, then concentrated by centrifugation. This treatment was
repeated three times, in order to completely eliminate the scaffolding
and the minor proteins.
Concentrated stocks of purified empty
procapsids, which contained exclusively coat protein, were treated with
GdmCl at a final concentration of 4.6 M overnight at room
temperature. This procedure dissociates the shells, liberating free
denatured coat protein molecules. These were extensively dialyzed in
experimental buffer (20 mM potassium phosphate, 25 mM NaCl, pH 7.6). Protein concentration was determined using
extinction coefficients at 280 nm of 1.04 for the wild-type, A108V,
T294I, and F353L subunits. An extinction coefficient of 0.907,
calculated following Edelhoch(1967), was used for W48Q, which lacks one
tryptophan. The protein solutions were always maintained at
temperatures between 4 and 10 °C after dialysis, and all
experiments were carried out with dialyzed protein samples that were
not older than 4 days. Dialyzed samples were used for DSC and some of
the circular dichroism (CD) experiments without dilution. They were
diluted 10-fold with experimental buffer for fluorimetry, and for the
CD studies in which a 1-cm cuvette was used.
For the gel
permeation chromatography (GPC) experiments with F353L in which
subsequent analysis by SDS-PAGE was performed, 90 µl of sample were
injected into the column. Four-drop fractions were collected at the
outlet and mixed with SDS-sample buffer lacking
In order to determine the apparent molecular weight of the species
detected by GPC, the column was calibrated with molecular weight
markers within the range 29,000-100,000. The excluded volume was
calibrated with blue dextran.
Far UV-CD
spectroscopy was used to monitor the secondary structure of the
wild-type and mutant proteins. An Aviv 60DS spectropolarimeter (Aviv
Associates, Lakewood, NJ) with a temperature control accessory
(Hewlett-Packard, Palo Alto, CA) was used. Spectra were recorded in
1-mm or 1-cm cells at protein concentrations of approximately 0.6 or
0.06 mg/ml, respectively. CD spectra were recorded from 190 to 350 nm,
each nm, with an averaging time of 1.5 s. For both fluorescence and CD,
samples were incubated for at least 10 min at 10 °C before the
spectra were taken.
Within infected cells at permissive temperature, both
wild-type and tsf mutant polypeptide chains fold into
assembly-competent species, which interact with scaffolding subunits
and minor proteins to form procapsids. Procapsid particles, assembled
at permissive temperature, either from wild-type coat protein or one of
the four tsf mutants (W48Q, A108V, T294I, and F353L), were purified
from appropriately infected host cells as described under
``Experimental Procedures.'' After extraction of scaffolding
and minor proteins, empty procap-sid shells containing only coat
protein were purified and then dissociated in 4.6 M GdmCl,
yielding denatured chains (Teschke and King, 1993). The denatured
chains were extensively dialyzed at 4 °C against experimental
buffer. In the absence of scaffolding protein and at the relatively low
protein concentrations used, we anticipated that the resulting
renatured coat protein subunits would form low levels of coat shells
and large aggregates (Prevelige et al., 1988).
If the mechanism of in
vivo temperature sensitivity were destabilization of folded
subunits, the tsf mutants would be expected to have significantly lower
melting temperatures than the wild-type protein. The tsf mutant
proteins were subjected to DSC in the same experimental conditions of
protein concentration and buffer as the wild-type protein. Thermal
denaturation was irreversible, and heated samples were visibly turbid.
The tsf mutant protein subunits have melting temperatures very close to
the wild-type (; Fig. 2), with three of the four
mutants having higher melting temperatures. A108V had a melting point 3
°C lower than wild-type protein. These data indicate that the in vivo mechanism of temperature sensitivity is not
destabilization of folded subunits. The enthalpies of denaturation
found for the tsf mutants were about half of the wild-type value (). This is consistent with fewer intramolecular
interactions in the folded state.
Since mutant fluorescence intensities were not just intermediate
between folded and unfolded protein, differences in properties between
the mutant and wild-type protein cannot be simply due to a reduced
yield of refolded mutant subunits.
The apparent molecular weights of
the two major peaks detected by GPC with the mutant protein samples
were calculated based on the elution volumes of protein standards. The
values obtained (approximately 44,000 and 92,000) are near those
predicted from the sequence for a coat monomer (47, 0) and dimer(94, 0) , respectively.
GPC at several temperatures was performed to determine if the two
species were in equilibrium with each other. As the temperature was
increased from 6 to 28 °C (Fig. 7), the mutant dimer peak
decreased in intensity and the monomer peak increased correspondingly.
The monomer and dimer peaks could be resolved from each other, and the
relative peak intensities were temperature-dependent, indicating that
these two species were undergoing slow equilibration.
The thermal stability of empty procapsids composed
exclusively of wild-type coat protein has been investigated recently
(Galisteo and King, 1993). This permits comparison of the stability of
the same protein in two different states: a folded but unpolymerized
state and a polymerized state forming the shell lattice. The DSC
transition corresponding to coat protein denaturation within the shell
lattice has a T
Similar results have
also been found using hydrostatic pressure to denature P22 procapsid
shells and subunits (Prevelige et al., 1994). The polymerized
shell was very stable to applied pressure, resisting dissociation up to
2.5 kilobars. In contrast, the soluble subunits were very unstable to
pressure, with denaturation beginning at pressures as low as 0.5
kilobar.
In contrast, the thermal stabilities of striated muscle
have been studied and do not exhibit extensive stabilization upon
oligomerization. In the case of actin, a stabilization of 10 °C in
the polymerized state has been reported (Bertazzon and Tsong, 1990),
while in case of myosin, no appreciable change in T
Extrapolating the DSC results to in vivo conditions would suggest that folded wild-type subunits are not
stable in the cell at 38 °C (Fig. 3). However, wild-type coat
protein assembles efficiently into shells in vivo at this
temperature (King et al., 1973; Gordon and King, 1993). Three
aspects of the in vivo process can account for this. First,
differences in cellular conditions as compared to our in vitro buffer may explain this discrepancy. For example, the presence of
molecular chaperones may increase the stability of folded coat protein in vivo (Martin et al., 1992). Second, in the cell
the coat protein assembles into procapsid shells with the aid of the
P22-encoded scaffolding protein, which may recognize a partially folded
intermediate. This would obviate the need for stable unpolymerized
folded subunits. In vitro, however, procapsid-like particles
can be assembled from folded coat and scaffolding subunits (Prevelige et al., 1988). Third, if assembly in vivo does
proceed from folded subunits, it may be that the rate of assembly is
much faster than the rate of subunit denaturation.
The stability of
polymerized forms of the T4 coat protein gp23 has been analyzed by DSC
by Steven and co-workers (Ross et al., 1985; Steven et
al., 1992). All of the polymerized forms had melting temperatures
significantly higher than the upper temperature limit for bacterial
growth. No cooperative transition was found for unpolymerized gp23,
consistent with marginal stability of the soluble subunits. The T4 coat
protein requires chaperones for formation of competent subunits within
cells (Coppo et al., 1973; van der Vies et al.,
1994).
Teschke and King(1993)
identified a kinetic intermediate in the refolding of the wild-type
coat protein, distinguished from the native state in its intrinsic
fluorescence and its binding of bis-1-anilino-8-naphthalenesulfonate.
Teschke and King(1995) have examined the kinetics of refolding of tsf
mutant proteins. They found that the tsf mutant and wild-type coat
proteins refolded with similar kinetics and through a similar kinetic
intermediate. Also, upon rapid dilution of the denatured chains out of
GdmCl, the mutants formed multimers, in accord with results presented
above.
Temperature-sensitive folding mutations have been
characterized extensively for the thermostable tailspike of P22
(Goldenberg and King, 1981; Goldenberg et al., 1983;
Sturtevant et al., 1989). The tsf amino acid substitutions had
no effect on the stability or activity of the tailspike, once matured
at permissive temperature. However, if expressed at restrictive
temperature, a folding intermediate was unable to continue productive
folding. Other temperature-sensitive folding mutants have been
described for D-lactate dehydrogenase (Truong et al.,
1991), luciferase (Sugihara and Baldwin, 1988), and interleukin-1
In vivo, the tsf mutant chains are rescued by
overexpression of the GroEL chaperone and can be recovered in a complex
with GroEL (Gordon et al., 1994). It will be important to
examine the in vitro refolding of the tsf coat chains in the
presence of the GroEL/S chaperone. It has been postulated that
chaperones maintain simian virus 40 coat protein pentamers in an
assembly-competent, incompletely folded form (Liddington et
al., 1991).
Several viruses assemble via small multimers of
coat protein molecules. The shells of RNA plant viruses (Sorger et
al., 1986; Larson et al., 1993) are formed from
polymerization of coat protein dimers, one of which is covalently
linked, around viral RNA (Stockley et al., 1986). The
adenovirus major coat protein forms trimers, in a process mediated by a
virus-encoded ``100K'' chaperone protein, prior to procapsid
shell assembly (Cepko and Sharp, 1982). Polyoma viruses assemble from
coat protein pentamers (Salunke et al., 1986), and
picornaviruses assembly proceeds via substructures composed of five
protomers of three different coat protein molecules (Rueckert, 1991).
Scan
rate was 2.0 K/min. T
Temperature was 10 °C.
We thank Marcos Milla for helpful guidance on circular
dichroism and R. T. Sauer for access to the instrument.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 87 °C,
H
1700 kJ/mol) (Galisteo, M. L., and King, J.(1993) Biophys. J. 65, 227-235) indicates that protein-protein
interactions within the capsid provide enormous stabilization. The
melting temperatures of the subunits carrying tsf substitutions were
similar to wild-type. At low temperatures, the tsf mutants, but not the
wild-type, formed non-covalent dimers, which were dissociated at
temperatures above 30 °C. Spectroscopic and calorimetric studies
indicated that the mutant proteins have reduced amounts of ordered
structure at low temperature, as compared to the wild-type protein.
Although complex, the in vitro phenotypes are consistent with
the in vivo finding that the mutants are defective in folding,
rather than subunit stability. These results suggest a role for
incompletely folded subunits as precursors in viral capsid assembly,
providing a mechanism of reaching multiple conformations in the
polymerized form.
Figure 1:
Schematic
pathway of bacteriophage P22 assembly.
P22 coat protein is 430 amino acids in length (Eppler et
al., 1991). 420 molecules of coat protein, about 300 molecules of
scaffolding protein, and other minor proteins co-assemble to form the
P22 procapsid particle (Fig. 1) (King et al., 1973; King
and Casjens, 1974; Fuller and King, 1982). P22 structural proteins are
not proteolytically processed during the assembly process, which
facilitates their study (Prevelige et al., 1988). In addition,
assembly-competent phage P22 coat protein subunits can be obtained in vitro, which permits comparison of the properties of
polymerized and unpolymerized protein (Fuller and King, 1981, 1982).
The formation of procapsids containing coat protein, scaffolding
protein, and one minor protein has been reproduced in vitro from purified proteins (Prevelige et al., 1988; Thomas
and Prevelige, 1991). The rate of procapsid formation in vitro showed a fifth order dependence on the concentration of coat
protein, which suggested that a coat protein pentamer forms a
rate-limiting initiation complex (Prevelige et al., 1993). The
P22 coat protein has been reported to be monomeric in purified form at
concentrations lower than 1.3 mg/ml (Prevelige et al., 1988).
(
)mutants (W48Q,
A108V, T294I, and F353L) in vitro, using DSC, column
chromatography, and spectroscopic techniques. We anticipated that study
of mutants from different regions of the coat protein could define the
importance of specific coat domains, and that in vitro studies
of mutants with different in vivo restrictive growth
temperatures would address the link between in vitro stability
and in vivo folding. In addition, two of the four mutations
involved aromatic residues, which are generally highly conserved. The
results presented in this report, in conjunction with previous studies,
point to the importance of elucidating the interplay between chain
folding, subunit stability, and subunit assembly.
Materials
All chemicals used were reagent grade.
All bacterial strains were derivatives of Salmonella typhimurium LT2. The suppressor minus host DB7136 and its suppressor plus
derivative DB7155 have been described by Winston et al. (1979).
Protein Purification and Sample
Preparation
Procapsids from cells infected with phage carrying
the wild-type gene 5 and from cells infected with phage
carrying the tsf alleles W48Q, A108V, T294I, and F353L were purified
essentially as described by Galisteo and King(1993). Infections were
carried out at 26.5 °C, which is a permissive temperature for the
tsf mutants. Host DB7136 cells were infected with phage strains
carrying the coat protein allele of interest, as well as gene 3
amber and gene 13 amber alleles, which block the phage
assembly process at the procapsid stage (King et al., 1973).
Gel Permeation Chromatography
A KNK-500-A series
liquid chromatograph (Konik Instruments) was used. About 10-20
µl of protein samples previously dialyzed in experimental buffer,
and within the range 0.5-0.7 mg/ml, were injected into a
Spherogel TSK-3000PWHR column (Beckman) equilibrated at several
temperatures. The flow rate was 0.5 ml/min. Absorbance at 280 nm was
registered continuously with an ERC-7211 UV detector (ERMA Inc., Tokyo,
Japan). Data were recorded in a graphic register and then digitized
with a Gentizier tablet model GT-1812D (Autocad, Autodesk). Overlapping
peaks were deconvoluted into two Lorentzian curves, and their elution
volumes and areas were determined, using the program PEAKS from Dr. E.
Freire (Johns Hopkins University, Baltimore, MD).
-mercaptoethanol.
Differential Scanning Calorimetry
DSC of protein
samples was carried out as described by Galisteo and King(1993). A
DASM-4 microcalorimeter (Mashpriborintorg, Moscow region, Russia) was
used. Protein concentrations were within the range 0.3-1.2 mg/ml.
Second scans of previously heated samples gave no thermal effects. All
samples were heated to 100 °C; in the wild-type sample alone, an
additional small thermal effect was observed at about 85 °C (due to
the existence of coat protein shells formed during the renaturation
process at low yield, which denature at that temperature).
Fluorescence and Circular Dichroism
Studies
Fluorescence spectra were recorded on a Hitachi F4500
fluorescence spectrophotometer, and the temperature was controlled with
a circulating water bath. The excitation wavelength was 280 nm.
Emission spectra were taken from 300 to 450 nm, at 240 nm/min, with an
excitation slit of 2.5 nm and an emission slit of 5 nm.
Thermal Denaturation of Wild-type and Mutant Proteins
Fig. 2shows the excess heat capacity versus temperature profiles for the soluble subunits, as determined by
differential scanning calorimetry. The DSC transition corresponding to
the thermal denaturation of wild-type coat protein was irreversible and
scan rate-dependent. The T was 40.0
°C at 2.0 K/min, and 38.0 °C at 0.5 K/min. The whole
transition, not just the T
, was shifted
to lower temperature at the lower scan rate (not shown). This suggests
that the DSC transition was completely determined by the kinetics of
the irreversible step(s). The subunits denatured rapidly at
physiological temperatures (Fig. 3). The half-time of
denaturation was
10 min at 38 °C. Electron microscopy
performed on heated samples, which had become visibly turbid,
demonstrated that the denatured protein had aggregated (not shown).
Figure 2:
Thermal stability of wild-type and mutant
proteins followed by DSC. Scan rate: 2.0 K/min. Protein concentration
was within the range 0.6-1.2 mg/ml.
Figure 3:
Wild-type P22 coat protein irreversible
denaturation is kinetically controlled. Aliquots (1 ml) of wild-type
protein solution at 0.61 mg/ml were placed in a water bath at 38 °C
for the indicated times (in minutes). After those times the samples
were cooled to 4 °C and subjected to DSC at 2.0 K/min. The bar corresponds to 20 kJ/Kmol.
The denaturation enthalpy of wild-type coat protein was 600
kJ/mol at 40.0 °C, the specific enthalpy of denaturation being 2.8
cal/g. This value is within the range found for typical compact
globular proteins that denature in this temperature range (Privalov,
1979) but is toward the low end of this range, which is consistent with
the protein not being highly compact.
Spectroscopic Properties of Wild-type and Mutant
Proteins
The secondary structures of purified wild-type and
mutant proteins were compared using circular dichroism spectroscopy at
10 °C (Fig. 4). The wild-type protein has a CD spectrum
typical of a protein that contains both -helix and
-sheet,
with minima at 221 and 209 nm (). The four mutant proteins
share similar CD spectra: a decreased signal at 221 nm and a shift of
the second minimum to 203 nm, both of which are indicative of a lower
content of ordered structure in these samples (Greenfield and Fasman,
1969).
Figure 4:
Far UV-CD spectra of wild-type and tsf
mutant proteins. Protein concentration was within the range
7.5-9.4 µM. Temperature was 10 °C.
, wild-type; --, W48Q; - - -, A108V; -
-, T294I; -
-, F353L. Spectra shown are averages of
five scans.
Fluorescence of wild-type and mutant proteins was carried out
at 10 °C (Fig. 5). Wild-type and three of the mutant proteins
have 6 tryptophanyl residues; the mutant W48Q has 5. They are
distributed throughout the amino acid sequence, so the fluorescence
emission spectrum yields information pertaining to the average
environment of these tryptophanyl residues. The wild-type spectrum has
its maximum emission intensity at 340 nm, indicating partial exposure
of tryptophanyl residues to the solvent in the native protein
(Lacowicz, 1983). The maximum emission wavelength is red-shifted for
the mutants with respect to wild-type (), indicating
increased overall exposure of tryptophanyl residues to solvent in these
proteins (Lacowicz, 1983). The intensity of the emission maximum was
larger for the mutants than the wild-type protein.
Figure 5:
Fluorescence spectra of wild-type and tsf
mutant proteins. Excitation was at 280 nm. Protein concentration was
1 µM. Temperature was 10 °C. Fluorescence raw
data have been divided by the respective protein concentrations and
normalized to the wild-type intensity at 340 nm (100%).
, wild-type; --, W48Q; - - -, A108V; -
-, T294I; -
-, F353L.
Wild-type and tsf
mutant proteins, denatured in 4.6 M GdmCl, had spectra with
maxima at 352 nm and fluorescence intensities 3-4 times lower
than in the absence of GdmCl (not shown). A similar result for the
wild-type protein in Tris buffer has been reported (Teschke and King,
1993). The red-shifted spectrum seen for the mutant subunits was
consistent with these subunits adopting a less ordered structure. Since
the unfolded wild-type protein gives a reduction in fluorescence
intensity, the increase in intensity seen for the folded tsf mutant
subunits was surprising. The mutants may specifically expose some
tryptophanyl residue(s) to solvent that were quenched by nearby amino
acids in the wild-type. Alternatively, the mutants could bury some
specific tryptophanyl residue(s), quenched by solvent or nearby amino
acids in the wild-type protein, while still giving an average overall
increase in exposure of tryptophanyl residues to solvent. The altered
association properties described below can account for such results.
Hydrodynamic Properties of Wild-type and Mutant Proteins
at Different Temperatures
GPC was utilized to examine the
conformational state of the proteins and to determine whether or not
the samples were heterogeneous. Fig. 6shows the GPC results for
the wild-type protein and the tsf mutant proteins at 14 °C. The
prominent peak observed in the wild-type sample eluted at the apparent
molecular weight expected for a globular monomer (V= 6.72 ± 0.02 ml). A
small peak in the void volume, due to the presence of coat protein
shells formed during the renaturation process, was also observed. The
nature of the small peak at V
=
8.1 ml is at present unclear.
Figure 6:
Gel permeation chromatography of wild-type
and tsf mutant proteins at 14 °C. Protein concentrations were
within the range 0.63-0.75 mg/ml. Raw data have been normalized
to protein concentration.
The mutant protein profiles contained,
in addition to the monomer peak, a second larger peak with an elution
volume very near that expected for a dimer (V = 5.98 ± 0.03 ml). The wild-type sample had a
small peak at this position. Small peaks representing higher multimers
were also present, especially in A108V. The A108V multimers eluted with
a V
= 5.33 ± 0.02 ml,
slightly slower than blue dextran.
Figure 7:
Gel permeation chromatography of tsf
mutants at the indicated temperatures. Protein concentrations for the
mutants W48Q, A108V, T294I, and F353L were, in mg/ml, 0.63, 0.66, 0.68,
and 0.72, respectively.
In the GPC
profile of the wild-type protein at 14 °C, a small peak with the
molecular weight of a dimer was present (Fig. 6). However, this
species was not in equilibrium with monomers, since its population did
not vary with temperature (not shown). P22 coat protein has one
cysteine residue per molecule (Cys-405), so we considered the
possibility that the wild-type dimeric species was disulfide-linked.
SDS-PAGE of the wild-type and mutant samples was performed in the
absence of -mercaptoethanol. Fig. 8shows that, in all
cases, a small population of disulfide-linked dimers exists. The
disulfide-linked dimers eluted from the GPC column with the
non-covalent dimers (Fig. 9), which confirmed our identification
of the dimer peak. Disulfide-linked coat protein molecules were not
present in the originally purified coat protein shells (not shown).
This was expected, since the bacterial cytoplasm in which the coat
protein molecules fold and assemble is a reducing environment.
Renaturation of F353L in the presence of DTT yielded exclusively
non-covalent dimers (Fig. 9).
Figure 8:
SDS-PAGE of wild-type and mutant coat
proteins. Pc, P22 purified wild-type procapsids; the major
band corresponds to the coat protein (47 kDa). Ph, purified
phosphorylase b (94 kDa). There are three lanes for every
protein allele (from left to right): purified empty
procapsid shells (Sh), coat protein subunits in Laemmli sample
buffer (+), and in the same buffer lacking -mercaptoethanol
(-). Samples were run on a 10% gel and
silver-stained.
Figure 9:
Non-covalent dimers still form in a
reducing environment. F353L was first renatured and then
chromotographed at 14 °C. Both processes were either in the
presence (top) or absence (bottom) of 10 mM DTT. Protein was at 0.75 mg/ml in the presence of DTT and 0.61
mg/ml in the absence of DTT. Aliquots collected at the outlet of the
column were mixed with Laemmli sample buffer without
-mercaptoethanol and run in 10% gels that were silver-stained. The arrow shows the position of a 96-kDa marker (phosphorylase b), which has the same molecular mass as a coat protein
dimer.
Analysis of Wild-type Coat Protein Stability
Comparison of Unpolymerized Coat Protein Stability with
That of Polymerized Subunits
The protein capsids of DNA viruses
are characterized by high stabilities, presumably evolved for the
protection of the genome during the extracellular stages of the life
cycle. However, the assembly of these shells is believed to be
associated with flexibility and switching between a variety of
conformations (Caspar, 1980; Caspar and Klug, 1962; Ross et
al., 1985; Prevelige et al., 1988, 1993; Steven et
al., 1992).
of 87 °C and a
calorimetric enthalpy of
1700 kJ/mol (Galisteo and King, 1993).
Unpolymerized wild-type coat protein subunits had a T
of 40 °C and a calorimetric
enthalpy of
600 kJ/mol (). This indicates that
protein-protein interactions within the shell lattice produce an
enormous stabilization of the coat protein subunits, increasing the T
by almost 50 °C. Presumably,
potential bonding interactions in the monomers that are not saturated
are used to make new contacts in the shells.
was observed (Bertazzon and Tsong,
1989).
The Denaturation of Unpolymerized Subunits Is under
Kinetic Control
The DSC transition corresponding to wild-type
protein denaturation was irreversible, and the calorimetric/van't
Hoff enthalpy ratio was very close to 1. This ratio was consistent with
the transition occurring at equilibrium and being two-state, followed
by an irreversible process at higher temperatures (Brandts et
al., 1989). However, the DSC transition was scan rate-dependent,
which indicates that it is under kinetic control (Galisteo et
al., 1991; Sánchez-Ruiz, 1992). This was confirmed by the
finding that the denaturation enthalpy decreased with time of
preincubation at 38 °C (Fig. 3). Kinetically controlled
denaturation precludes application of equilibrium thermodynamic
analysis (Sánchez-Ruiz, 1992).
Unpolymerized Coat Protein Subunits Are Not Stable at
Physiological Temperatures
The wild-type coat protein monomers
have both a low melting point and a relatively low specific enthalpy of
denaturation, suggesting that they are not tightly locked into a folded
conformation. This is consistent with the necessity for viral coat
proteins to adopt a variety of conformations and, therefore, to be
structurally flexible (Caspar and Klug, 1962; Harrison, 1984; Prasad et al., 1993).
Analysis of tsf Mutant Coat Protein Stability
The tsf Substitutions Do Not Destabilize Renatured
Subunits
In vivo, the W48Q, A108V, T294I, F353L, and
wild-type coat protein alleles had restrictive growth temperatures of
about 41, 39, 33, 37, and >42 °C, respectively (Gordon et
al., 1994). In vitro, soluble wild-type subunits were not
significantly more stable than mutant subunits (). The
subunits were essentially monomeric in this temperature range (Fig. 7). The DSC results support the idea that the
mutants' temperature sensitivity is due to destabilization at
elevated temperatures of a folding intermediate occurring prior to the
formation of the ``folded'' species subjected to DSC. In this
work, the subunits studied were generated by dialysis out of GdmCl at 4
°C. Presumably, in vivo a folding intermediate which is
present at restrictive temperatures is destabilized by the tsf
mutations, thereby leading to inclusion body formation. In agreement
with the DSC results, pressure dissociation studies have shown that the
tsf W48Q and G232D mutant subunits had stabilities similar to wild-type
subunits (Foguel et al., 1995).
(Chrunyk et al., 1993).
Mutant Dimer Formation and the Pathway of Viral
Assembly
Spectroscopic experiments suggested that the mutants
have less compact and more flexible conformations than the wild-type
protein. This suggests that the non-covalent dimers form preferentially
from partially folded molecules. In vitro, the tsf mutants
could increase the likelihood of dimerization by prolonging the
lifetime of folding intermediates from which the dimers are formed. The
non-covalent dimers could be true assembly intermediates and contain
protein-protein contacts found at 2-fold interfaces on the viral shell.
An alternative model is that once the subunits have achieved a subset
of the T = 7 conformations, for example those found at
dimer interfaces, they are no longer competent for further
polymerization. In this model, competence for shell formation requires
a partially folded monomeric intermediate, able to switch into the
different quasi-equivalent conformations needed to form a closed shell.
Recent results of Teschke and King(1995) are consistent with the second
model.
The tsf Coat Protein Mutants Behave Similarly
The
three-dimensional structure of the P22 coat protein has not yet been
determined. Structural studies of this protein in unpolymerized form
are difficult since protein solutions at concentrations higher than 1.3
mg/ml induce strong protein aggregation (Prevelige et al.,
1988), thereby preventing structural studies by Fourier transform
infrared, NMR, or x-ray crystallography. Thus, the location of the tsf
mutant residues in the folded protein is unknown. One possible
explanation for the similar behavior of all four mutants is that the
mutated residues may lie close to each other in the tertiary structure,
and that region of the protein would be important for the correct
folding of the whole structure. Another interpretation is that the
individual mutations may identify different sites important for coat
protein folding. Either model is consistent with the altered forms
being substrates for chaperone recognition within cells.
Table: Transition midpoints (T) and
denaturation enthalpy values (
H) for the thermal denaturation of
the P22 coat wild-type and tsf mutant proteins obtained by DSC
values are in
°C and
H values in kJ/mol. Protein concentration was
within the range 0.6-1.2 mg/ml. Parameters were obtained from at
least three independent experiments. The standard deviations of the
measurements are also shown.
Table: Spectroscopic parameters of the wild-type and
tsf mutants in 20 mM phosphate, 25 mM NaCl, pH 7.6
(nm) corresponds to
the minimum intensities in the far-UV CD spectra for the wild-type and
tsf mutant coat proteins.
(nm) corresponds to the
maximum intensity in the emission fluorescence spectra for the
indicated proteins.
H, calorimetric enthalpy; PAGE, polyacrylamide gel
electrophoresis; T, temperature of the maximum heat capacity; V, elution volume.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.