©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stability of Wild-type and Temperature-sensitive Protein Subunits of the Phage P22 Capsid (*)

Mara L. Galisteo (1) (2), Carl L. Gordon (2), Jonathan King (2)(§)

From the (1)Departamento de Qumica Fsica, Facultad de Ciencias, Universidad de Granada, Granada 18071, Spain and the (2)Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 = 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.


INTRODUCTION

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).


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).

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()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.


EXPERIMENTAL PROCEDURES

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).

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.

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).

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 -mercaptoethanol.

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.

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.

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.


RESULTS

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).

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.

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.

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.

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.

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.

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.


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.




DISCUSSION

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).

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 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.

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 Twas 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).

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).

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).

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 (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.

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).

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

Scan rate was 2.0 K/min. T 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

Temperature was 10 °C. (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.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM17980 (to J. K.), a Howard Hughes predoctoral fellowship (to C. G.), and Spanish Ministry of Education and Science Grant PB90-0876. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Dept. of Biology, 68-330, Massachusetts Institute of Technology, Cambridge, MA 02139. Tel.: 617-253-4700; Fax: 617-253-8699.

The abbreviations used are: tsf, temperature-sensitive folding; CD, circular dichroism; DSC, differential scanning calorimetry; DTT, dithiothreitol; GPC, gel permeation chromatography; GdmCl, guanidinium chloride; H, calorimetric enthalpy; PAGE, polyacrylamide gel electrophoresis; T, temperature of the maximum heat capacity; V, elution volume.


ACKNOWLEDGEMENTS

We thank Marcos Milla for helpful guidance on circular dichroism and R. T. Sauer for access to the instrument.


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