Báo cáo khoa học: Dissecting the role of protein–protein and protein–nucleic acid interactions in MS2 bacteriophage stability potx

Dissecting the role of protein–protein and protein–nucleic
acid interactions in MS2 bacteriophage stability
Sheila M. B. Lima
1
, Ana Carolina Q. Vaz
1
, Theo L. F. Souza
1
, David S. Peabody
2
, Jerson L. Silva
1
and Andre
´
a C. Oliveira
1
1 Programa de Biologia Estrutural and Centro Nacional de Ressona
ˆ
ncia Magne
´
tica Nuclear de Macromole
´
culas, Instituto de Bioquı
´
mica
Me
´
dica, Universidade Federal do Rio de Janeiro, Brazil
2 Department of Molecular Genetics and Microbiology and Cancer Research and Treatment Center, University of New Mexico School of
Medicine, Albuquerque, NM, USA
Specific protein–protein and protein–nucleic acid inter-

actions are required for successful assembly of a large
variety of biologically important macromolecular com-
plexes, including viruses. We used the bacteriophage
MS2 as a model for the study of such interactions.
MS2 is a member of a large group of small RNA
phages that infect Escherichia coli [1]. Its icosahedral
shell consists of 180 copies of coat protein (M
r
13 728)
arranged in a T ¼ 3 quasi-equivalent surface lattice
surrounding the ssRNA genome. Each virion also con-
tains one copy of the maturase (or A) protein, respon-
sible for attachment of the virus to E. coli through the
F-pilus. Coat protein folds as a dimer of identical sub-
units and consists of a 10-stranded antiparallel b-sheet
facing the interior of the phage particle, with antiparal-
lel, interdigitating a-helical segments on the virus’
Keywords
coat protein interactions; fluorescence;
hydrostatic pressure; MS2 bacteriophage;
virus-like particles
Correspondence
J. L. Silva and A. C. Oliveira, Avenida
Bauhinia, 400 – CCS ⁄ ICB ⁄ Bl E, sl. 08,
Cidade Universita
´
ria, CEP, Rio de Janeiro,
RJ 21941-590, Brazil
Fax: +55 21 3881 4155
Tel: +55 21 2562 6756

E-mail:
E-mail:
(Received 2 August 2005, revised 26 January
2006, accepted 6 February 2006)
doi:10.1111/j.1742-4658.2006.05167.x
To investigate the role of protein–protein and protein–nucleic acid interac-
tions in virus assembly, we compared the stabilities of native bacteriophage
MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assem-
bly-defective coat protein mutant (dlFG) and its single-chain variant
(sc-dlFG). Physical (high pressure) and chemical (urea and guanidine
hydrochloride) agents were used to promote virus disassembly and protein
denaturation, and the changes in virus and protein structure were moni-
tored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluor-
escence, and light scattering. We found that VLPs dissociate into capsid
proteins that remain folded and more stable than the proteins dissociated
from authentic particles. The proposed model is that the capsid disassem-
bles but the protein remains bound to the heterologous RNA encased by
VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids,
because it lacks the 15-amino acid FG loop involved in inter-dimer inter-
actions at the viral fivefold and quasi-sixfold axes. This protein was very
unstable and, when compared with the dissociation ⁄ denaturation of the
VLPs and the wild-type virus, it was much more susceptible to chemical
and physical perturbation. Genetic fusion of the two subunits of the dimer
in the single-chain dimer sc-dlFG stabilized the protein, as did the presence
of 34-bp poly(GC) DNA. These studies reveal mechanisms by which inter-
actions in the capsid lattice can be sufficiently stable and specific to ensure
assembly, and they shed light on the processes that lead to the formation
of infectious viral particles.
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonate; GdnHCl, guanidine hydrochloride; VLP, virus-like particle.

FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1463
outer surface. The 15-residue FG loop extends
from both ends of the dimer and is responsible for
many of the interdimer contacts essential for virus
assembly [2].
Coat protein acts both as a translational repressor
and as the principal viral structural protein, interacting
specifically with a 19-nucleotide RNA hairpin to
repress translation of the viral replicase and to nucle-
ate assembly of the viral shell on genome RNA [3–7].
It has been extensively studied using genetic approa-
ches, and a large number of mutational variants
affected in their protein–RNA and protein–protein
interactions have been produced [8,9]. We recently des-
cribed a comparative study with three ts mutants of
bacteriophage MS2, in which in each mutant only one
coat protein amino acid was substituted. Each substi-
tution was sufficient to change the stability of viral
capsid in the presence of denaturing agents [10]. Here
we describe studies of additional coat protein variants.
As part of a study to define which interactions are
important for virus stability, we compared authentic
MS2 virions with virus-like particles (VLPs) produced
when coat protein is overexpressed in E. coli from a
plasmid [11]. To examine the stability of the coat pro-
tein dimer in the absence of the intersubunit interac-
tions found in the capsid, we used a mutant, named
dlFG, in which the FG loop, a 15-residue sequence
connecting the F and G b-strands, is replaced with
two amino acids sufficient to make a b-turn. Although

unable to form capsids, the dlFG dimer retains its abil-
ity to bind RNA [3,5,12–17]. We also determined the
effects on stability of genetic fusion of the two sub-
units of the dlFG dimer using the variant named
2CTdlFG, which takes advantage of the physical prox-
imity of the N-terminus and C-termius of the two
monomers to covalently link them. To facilitate
purification, both dlFG and a single-chain dimer,
sc-dlFG, contain an N-terminal six-histidine nickel-
affinity tag.
Using intrinsic fluorescence of Trp residues, extrinsic
fluorescence of the probe bis-8-anilinonaphthalene-1-
sulfonate (bis-ANS), light scattering, and CD, we
monitored conformational changes promoted by high
pressure and high concentrations of urea and guani-
dine. Concerning the tertiary structure, the relative
stabilities of the different forms are as follows:
dlFG < sc-dlFG < MS2 < VLP. The higher stability
of the capsid protein bound to heterologous nucleic acid
may serve as a ‘biological sieve’. In contrast, authentic
MS2 particles dissociate and unfold co-operatively,
which would guarantee that any particle without the
authentic RNA would be locked in a state lacking the
ability to release the RNA during infection.
Results
Dissociation and denaturation of wild-type
virus and VLPs induced by urea and guanidine
hydrochloride (GdnHCl)
Intrinsic fluorescence provides a convenient means to
monitor changes in protein conformation in the pres-

ence of denaturing agents. The tryptophan residues
buried in the hydrophobic interior of the protein
emit fluorescence when excited at 280 nm. When the
protein unfolds in the presence of denaturing agents,
the exposure of buried residues reflects the conform-
ational changes in the protein. The coat protein of
MS2 has two tryptophan residues, Trp32 and Trp82.
Trp32 clearly resides within the hydrophobic core of
the protein. The other residue, Trp82, is partially
solvent-exposed [17]. Its environment is determined
primarily by interactions within the dimer, not by
interactions between dimers. Thus, tryptophan fluo-
rescence should predominantly monitor dimer dena-
turation rather than capsid dissociation. Meanwhile,
light-scattering measurements are sensitive to the size
of the particle and can be used to monitor capsid
dissociation. The use of the two methods permitted
us to discriminate between the two phenomena inves-
tigated here, dissociation of the capsids and denatur-
ation of the coat protein.
The wild-type coat protein, when expressed in E. coli
from a plasmid spontaneously associates into capsids
and packages host RNAs, thus forming VLPs. We
treated VLPs and native virus with increasing concen-
trations of urea to compare the role of native RNA
with heterologous RNA in the stability of the capsid
protein.
Surprisingly, as measured by tryptophan fluores-
cence, VLPs appear to be substantially more stable
than virus particles themselves. The spectral center of

mass of VLPs did not change significantly until 5 m
urea, indicating that the capsid protein does not dena-
ture until this urea concentration is achieved (Fig. 1A).
MS2, however, begins to significantly unfold at around
3.5–4 m. This result is all the more surprising when
compared with the VLPs of other RNA viruses, which
are generally less stable than the authentic virus in the
presence of nonviral RNA [18]. Similar behavior was
observed in the presence of a different denaturant,
GdnHCl, as the VLP starts to denature only after
3.5 m GdnHCl (Fig. 1B). In fact, during both treat-
ments (5 m urea and 3.5 m GdnHCl), there were
minimal changes on solvent exposure of tryptophan
residues on VLP coat protein, compared with the other
forms.
Bacteriophage MS2 stability S. M. B. Lima et al.
1464 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
To check whether the stability was of the entire par-
ticle or of the capsid protein, light-scattering measure-
ments were performed for MS2 (Fig. 2A) and VLPs
(Fig. 2B). When we plotted the spectral center of mass
and light scattering for MS2 in the same plot, we
observed that the curves were superimposable, indica-
ting that virus disassembly was coincident with subunit
unfolding (Fig. 2A). For the VLPs, however, the
curves were different, suggesting that protein unfolding
occurs only after capsid disassembly (Fig. 2B).
Overall, the results indicate that authentic MS2
particles dissociate and denature co-operatively. In
contrast, VLPs disassemble at lower denaturant

concentration but denature at much higher urea con-
centrations than the MS2 capsid protein. We propose
that the VLP capsid disassembles but the protein
remains bound to the heterologous RNA encased by
the VLPs. The VLPs have been previously character-
ized [11] showing that VLP capsids contain a hetero-
geneous population of RNAs, with two predominant
species corresponding to about 1800 and 200 nucleo-
tides. Northern-blot analysis shows that the coat
protein encapsidates host RNAs, including species
derived from rRNA precursors. The average size of the
Fig. 1. Effects of urea and guanidine on the stability of bacterio-
phage MS2 wild-type, VLPs, dlFG and sc-dlFG. (A) Effects of
increasing urea concentrations on the spectral center of mass of
(d) MS2 WT, (n) VLPs, (m) dlFG, and (r) sc-dlFG. Incubation was
overnight at each concentration. (B) Effect of guanidine was meas-
ured by the spectral center of mass of tryptophan fluorescence
emission. The sample was excited at 280 nm and the emission
was measured at 300–420 nm. The buffer used was 10 m
M
Tris ⁄ HCl ⁄ 100 mM NaCl ⁄ 0.01 mM EDTA, pH 7.5.
Fig. 2. Comparison between spectral center of mass and light-scat-
tering values of bacteriophage MS2 (A) and VLPs (B) during urea-
induced dissociation ⁄ denaturation. To verify the dissociation and
denaturation processes, we compared in the same plot the spectral
center of mass and light-scattering measurements in the presence
of increasing concentrations of urea. (A) (d) Spectral center of
mass; (s) light scattering for MS2. (B) (n) Spectral center of mass;
(h) light scattering for VLPs. For the light-scattering measurements,
the sample was excited at 320 nm and the emission was meas-

ured at 315–325 nm. For the spectral center of mass, the sample
was excited at 280 nm and measured at 300–420 nm.
S. M. B. Lima et al. Bacteriophage MS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1465
heterologous RNA is much smaller than that of the
MS2 RNA. It is noteworthy that the two particles
have similar hydrodynamic behavior, and electron
microscopy shows that they are very similar (data not
shown).
To verify the urea-induced and GdnHCl-induced
changes in the secondary structure of the proteins, we
analyzed the UV CD spectra of MS2 and VLPs in the
absence and presence of 5.0 m and 9.0 m urea and
3.5 m and 5.0 m GdnHCl, the concentrations at which
the greatest difference between the samples was
observed. CD spectra were measured in the range 300–
190 nm and showed that the loss of secondary structure
probably accompanied the loss of tertiary structure.
The CD data reveal one negative peak at 216 nm,
corresponding to a high b-sheet content, and one pos-
itive peak around 260–270 nm contributed by RNA
[19]. In the presence of 5.0 m urea there was a decrease
of  70% of ellipticity at 216 nm for MS2, indicating
a substantial loss of secondary structure, and at 9 m
urea no residual secondary structure was present
(Fig. 3A). A similar result was obtained with GdnHCl
treatment (Fig. 3B). However, the VLPs showed few
changes in secondary structure in the presence of 5.0 m
urea, and no change in the presence of 3.5 m GdnHCl,
confirming its higher stability (Fig. 3C,D).

Denaturation of the assembly-defective coat
protein mutants (dlFG and sc-dlFG) induced by
urea and GdnHCl
The loop between the F and G b-strands (FG loop) of
the bacteriophage MS2 coat protein subunit forms
contacts between dimers important for capsid assem-
bly. Because it lacks this loop, the dlFG mutant can-
not form capsids, but instead is encountered in the
form of dimers [5,14]. To understand the importance
of capsid protein–protein contacts for the stability of
coat protein, we determined the stability of dlFG and
compared it with the stability of the genetically fused
coat protein dimer, sc-dlFG. We induced dissociation
of dlFG and sc-dlFG with increasing concentrations of
urea between 1 and 8 m (Fig. 1A). The data showed
significant differences between the stability of dlFG,
which begins to unfold at  1 m urea and unfolds
completely by 4 m, and authentic virions, whose
unfolding only begins at around 4 m and is not com-
plete until 6 m. The sc-dlFG single-chain dimer was
about as stable as the virus particle. The shift in the
spectral center of mass  1500 cm
)1
at 4 m urea sug-
gested complete denaturation of dlFG at this urea con-
centration. On the other hand, complete denaturation
of virus and sc-dlFG was observed only in the pres-
ence of 6 m urea, and even more urea was needed to
promote complete denaturation of the VLPs.
A similar instability profile was observed for dlFG

in the presence of GdnHCl, where 1.5 m was enough
for complete denaturation, but the virus was denatured
only with 3.5 m. Although the urea denaturation of
sc-dlFG was very similar to the virus, in GdnHCl it
denatured more easily (Fig. 1B). It should be noted
that the FG loop deletion removes Trp82, accounting
for the differences observed between the initial spectral
center of the mass of both dlFG and sc-dlFG com-
pared with wild-type virus and VLPs, which both
retain Trps.
CD studies were also performed with the assembly-
defective mutants. With dlFG the presence of 2.0 m
urea promoted  50% decrease in ellipticity at 216 nm,
and no secondary structure was detected in the pres-
ence of 5.0 m. The same behavior was observed in the
presence of 1.0 m and 3.5 m GdnHCl. The data
showed that the loss of secondary structure accompan-
ied the loss of the tertiary structure (Fig. 3E,F). Again,
sc-dlFG was substantially more stable to these denatu-
rants than dlFG (Fig. 3G,H).
Pressure-induced dissociation and denaturation
of wild-type virus, VLPs, dlFG and sc-dlFG
Pressure effects are governed by Le Chatelier’s princi-
ple, where an increase in pressure favors reduction of
the volume of a system, leading to dissociated forms.
A key advantage of hydrostatic pressure is that it does
not perturb the chemical composition of the solvents,
or the internal energy of the protein [2,20,21].
The samples of MS2 and VLPs were diluted to a
final concentration of 50 lgÆmL

)1
and incubated for
20 min under increasing pressures up to 3.4 kbar.
Hydrostatic pressure effects were monitored by
Fig. 3. UV CD spectra of MS2 wild-type, VLPs, dlFG and sc-dlFG. Conformational changes in the secondary structure of MS2 bacteriophage
and mutant coat protein. (A,C) Treated with 4.5
M urea (dashed line), 9 M urea (dotted line) and control (solid line). (B,D) Treated with 3 M
GdnHCl (dashed line), 5 M GdnHCl (dotted line) and control (solid line). (E,G) Treated with 2 M urea (dashed line) and 5 M urea (dotted line)
and control (solid line). (F,H) Treated with 1
M GdnHCl (dashed line), 3.5 M GdnHCl (dotted line) and control (solid line). The spectra were
obtained in 10 m
M Tris ⁄ HCl buffer, pH 7.0, using a 0.1-cm path length quartz cuvette. The samples were diluted to a final concentration of
100 lgÆmL
)1
. The spectropolarimeter used was a Jasco J-715 1505 model. Wavelength range 300–200 nm. The data are representative of
three experiments.
Bacteriophage MS2 stability S. M. B. Lima et al.
1466 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
S. M. B. Lima et al. Bacteriophage MS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1467
changes in the spectral center of mass and by light
scattering. In contrast with the effects of chemical
agents, we found that high pressure was able to pro-
mote only small changes in the spectral center of mass
of the MS2 virus (Fig. 4). VLPs again seemed more
stable than the virus, as we observed no significant
changes at all in the spectral center of mass (Fig. 4).
There were also no significant changes in light scatter-
ing for VLPs and MS2 particles.
To confirm that high pressure did not affect the

virus and VLPs, we analyzed the treated samples by
HPLC. The samples treated by pressure showed the
same behavior as the untreated ones, confirming that
pressure did not dissociate the viral particles (data not
shown).
We also investigated the denaturation of dlFG and
sc-dlFG under pressure. Structural changes in dlFG
were followed by a significant shift in the Trp emission
spectrum, indicating increasing exposure to the polar
solvent. The coat protein mutant dlFG was more sus-
ceptible to pressure than was the virus, VLPs, or
sc-dlFG, 1.8 kbar being sufficient to promote complete
denaturation (Fig. 4). For sc-dlFG, complete denatura-
tion was observed only after 2.5 kbar.
bis-ANS binding assay
During dissociation and denaturation processes, pro-
teins expose hydrophobic segments and acquire the
ability to bind certain hydrophobic probes. As part
of our characterization of chemical-induced and pres-
sure-induced denaturation, we used the fluorophore
bis-ANS. This probe binds noncovalently to non-
polar segments in proteins, especially in proximity to
positive charges [22]. Because its binding is accom-
panied by a large increase in its fluorescence quan-
tum yield, it is useful in following protein structural
changes, and has been used to monitor conforma-
tional changes in capsid proteins during virus disas-
sembly.
At atmospheric pressure and in the absence of urea
and GdnHCl, the MS2 bacteriophage and VLPs did

not bind bis-ANS, showing that these particles do not
present exposed hydrophobic segments. High urea and
GdnHCl concentrations did not promote significant
binding to any of the particles, suggesting that the
denaturation of the protein was essentially complete
and prevented probe binding (data not shown). How-
ever, for dlFG and sc-dlFG, the probe bound the pro-
tein in the absence of either denaturant, and at low
urea concentrations an increase occurred in bis-ANS
fluorescence emission, but when the urea concentration
was further increased, the probe became unbound. The
binding of bis-ANS to dlFG and sc-dlFG promoted a
shift in the urea denaturation curves, suggesting a sta-
bilizing effect of the probe (Fig. 5A). The same effect
was not observed for GdnHCl-induced denaturation,
and sc-dlFG was actually less stable in the presence of
the probe (Fig. 5B).
When wild-type MS2 and VLPs were submitted to
high pressure in the presence of bis-ANS, again no sig-
nificant binding was observed (data not shown). How-
ever, when dlFG was pressurized in the presence of the
probe, there was a sevenfold increase in its emission,
suggesting that under pressure the protein exposes
hydrophobic segments and retains some degree of sec-
ondary structure. Furthermore, the conformation of
the pressure-denatured state is different from that of
the urea-denatured state (Fig. 6A).
The denaturation of dlFG promoted by pressure
was measured in the presence and absence of bis-ANS,
and the probe showed the same protective effect

observed on urea-induced denaturation (Fig. 6B). It
should be noted, however, that in all assays conducted
in the presence of bis-ANS, the spectral center of mass
did not return to its initial value after pressure release,
and an increase in the light-scattering value was
observed (data not shown). This result suggests that
the coat protein dimer may be undergoing aggregation.
For sc-dlFG, again we observed that the binding of
the probe destabilized the protein, as it did in the pres-
ence of GdnHCl (Fig. 6B).
Fig. 4. Pressure stability of MS2 bacteriophage wild-type, VLPs,
dlFG and sc-dlFG. The effects of pressure on MS2, VLPs, dlFG and
sc-dlFG at room temperature were analyzed. The effect was meas-
ured by spectral center of mass of tryptophan fluorescence emis-
sion. (d) MS2 WT; (n) VLPs; (m) dlFG; (r) sc-dlFG. The samples
were excited at 280 nm, and the emission was measured at
300–420 nm. The buffer used was 10 m
M Tris ⁄ HCl ⁄ 100 mM
NaCl ⁄ 0.01 mM EDTA, pH 7.5. Incubation at each pressure was for
20 min.
Bacteriophage MS2 stability S. M. B. Lima et al.
1468 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
Role of the nucleic acid–protein complex in the
stability of dlFG
As shown before, pressure was able to completely
denature dlFG above 1.8 kbar. To compare the stabil-
ity of the protein in the presence of different nucleic
acids, we incubated the coat protein samples with
dsDNA [a nonspecific poly(GC) DNA], tRNA and the
RNA hairpin sequence of MS2 (ratio 2 : 1, pro-

tein ⁄ DNA or tRNA or RNA), and the pressure stabil-
ity was checked. DNA poly(GC) stabilized the coat
protein dimer. p
1 ⁄ 2
, the pressure necessary to promote
half denaturation, shifted to higher pressures when the
protein was mixed with DNA. On the other hand,
when we incubated the coat protein with tRNA or the
hairpin sequence, neither was able to protect the pro-
tein against denaturation (Fig. 7A). After pressure
release, the value of the spectral center of mass of
dlFG emission in the presence of DNA returned
almost completely to the initial value, but again we
observed an increase in light scattering, suggesting
Fig. 5. Urea and guanidine treatment of dlFG and sc-dlFG in the
presence of bis-ANS. (A) Effects of increasing urea concentrations
on the spectral center of mass of (m) dlFG and (r) sc-dlFG in the
absence of bis-ANS, and in the presence of 1 m
M bis-ANS (n) dlFG
and (e) sc-dlFG. Incubation was overnight at each concentration.
(B) Effects of increasing GdnHCl concentrations on the spectral
center of mass of (m) dlFG and (r) sc-dlFG in the absence of bis-
ANS and in the presence of 1 m
M bis-ANS for (n) dlFG and (e)
sc-dlFG. Incubation was overnight at each concentration. The effect
was measured by spectral center of mass of tryptophan fluores-
cence emission. The sample was excited at 280 nm, and the emis-
sion was measured at 300–420 nm.
Fig. 6. bis-ANS binding to coat protein dimer (dlFG) under pressure
and pressure stability of dlFG and sc-dlFG in the absence and pres-

ence of bis-ANS. (A) Structural changes in dlFG were analyzed by
fluorescence of the probe bis-ANS at a final concentration 1 l
M.
Excitation wavelength was 360 nm and emission wavelength range
400–600 nm. Inset: fluorescence emission spectra of bis-ANS dur-
ing the pressurization process. (B) Effect of pressure on the coat
protein in the presence of bis-ANS for (n) dlFG and (e) sc-dlFG
and in the absence of the probe for (m) dlFG and (r) sc-dlFG, was
analyzed at room temperature. The effect was measured by spec-
tral center of mass of tryptophan fluorescence emission. The sam-
ple was excited at 280 nm, and the emission was measured at
300–420 nm.
S. M. B. Lima et al. Bacteriophage MS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1469
that, like bis-ANS, this DNA promoted an aggregation
process. The assay in the presence of DNA and bis-
ANS in the same concentration (1 : 1 : 1) pro-
tein ⁄ DNA ⁄ bis-ANS, showed that the protein bound
bis-ANS, because there was an increase in the fluores-
cence emission of the probe when the protein was sub-
jected to high pressure, but the DNA could not
protect the protein dimer against dissociation. The
presence of both DNA and bis-ANS decreased the sta-
bility of the protein (Fig. 7B).
Effects of high temperature on the secondary
and tertiary structure of dlFG and sc-dlFG in the
absence and presence of bis-ANS
We confirmed the results observed when we submitted
sc-dlFG to high pressure and GdnHCl in the presence
of bis-ANS using high temperature. We subjected

dlFG and sc-dlFG to elevated temperatures in the
presence and absence of bis-ANS and analyzed the
changes by fluorescence emission (Fig. 8A) and far-UV
CD (Fig. 8B). The results confirmed that the probe
decreased the stability of sc-dlFG, as the temperature
necessary to start the denaturing process decreased by
almost 20 °C and for dlFG the presence of the probe
did not change the stability (data not shown).
Discussion
Assembly of infectious bacteriophage MS2 requires the
proper folding of coat protein dimers and their self-
assembly into an icosahedral capsid, while selectively
encapsulating a single copy of viral plus-strand RNA
by the coat, and ignoring viral minus strands and host
nucleic acids. Moreover, the particle must be stable
enough to survive outside the host, but not so stable
as to prevent the release of the viral genome when a
new susceptible host is encountered. The complex
formed between coat protein and RNA has been inves-
tigated [5,9,23,24], and amino acid–nucleotide inter-
actions contributing to its stability have been
characterized [13,25], but the role of protein–protein
and protein–RNA interactions in virus stability is not
completely understood, and we have investigated these
questions in this work. We sought to determine the
role of protein–RNA and protein–protein interactions
in virus stability, measuring the effects of urea,
GdnHCl and high pressure on the structure and stabil-
ity of whole particles (bacteriophage MS2 and VLPs)
and assembly-defective coat protein species [coat pro-

tein dimers (dlFG) and single-chain dimers (sc-dlFG)].
The coat protein of bacteriophage MS2 expressed in
E. coli forms intracellular VLPs that package a precur-
sor form of 16S rRNA, which happens to contain a
translational operator-like sequence near its 5¢ end
[11]. The results reported here show that VLPs and
MS2 behave differently when perturbed with denatur-
ing agents. Whereas authentic MS2 particles dissociate
and denature co-operatively, VLPs undergo disassem-
bly at lower denaturant concentration and denatura-
tion at much higher concentration than the MS2
Fig. 7. Comparison of stability of dlFG under pressure in the pres-
ence and absence of nucleic acids and bis-ANS binding assay on
dlFG in the presence and absence of DNA. (A) Effect of pressure
on the dlFG coat protein at room temperature was analyzed in the
absence of nucleic acids (m) and in the presence of 34-bp DNA
poly(GC) (.) and yeast tRNA (,). The effect was measured by
spectral center of mass of tryptophan fluorescence emission. The
sample concentration was 1 l
M as well as the DNA and tRNA. (B)
Effect of pressure on the dlFG coat protein was analyzed in the
absence of nucleic acids (m), in the presence of 34-bp DNA
poly(GC) (.), bis-ANS (n) and in the presence of both bis-ANS
(1 l
M) and DNA (1 lM)(
~
). The effect was measured by spectral
center of mass of tryptophan fluorescence emission. The sample
concentration was 1 l
M.

Bacteriophage MS2 stability S. M. B. Lima et al.
1470 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
capsid protein. The only feasible explanation is the for-
mation of complexes of folded capsid protein with the
small RNAs that compose the VLPs [11]. The presence
of maturase protein in authentic particles may play a
role in the different behavior. In the natural virion, a
part of the maturase protein is present on the inner
surface of the capsid shell where it interacts with viral
RNA. Another part must be present on the outside of
the shell where it is responsible for adsorption of the
virus to the cellular receptor, the F-pilus. During infec-
tion, maturase enters the cell together with the RNA
genome, leaving behind an intact capsid.
The dlFG version of coat protein is dimeric and
therefore lacks both the protein–protein and protein–
nucleic acid interactions that normally occur within
the capsid. Our results show that dlFG is very unstable
to denaturants when compared with MS2, VLP and
sc-dlFG (Fig. 1A,B). Further investigation is necessary
to understand the role of the FG loop in dimer stabil-
ity, by comparing the wild-type and dlFG mutant.
Here we demonstrate that the dimeric protein is more
susceptible to denaturation. We think this is probably
mostly due to the absence of the intersubunit contacts
normally present in the capsid. Although we cannot
rule out a contribution of the FG-loop itself to dimer
stability, previous studies with several viruses show
that, when the wild-type protein is isolated from the
capsid, it becomes more unstable to chemical and

physical denaturing agents [26–28].
Bis-ANS-binding assays during pressurization of
dlFG and sc-dlFG suggested the exposure of hydro-
phobic residues, as the fluorescence intensity of the
probe increased about sevenfold. However, in the pres-
ence of urea or GdnHCl, there is nonexpressive bis-
ANS binding, suggesting that the pressure-denatured
state is different from the chemical-denatured state.
Furthermore, the probe partially protected both forms
against urea (but not guanidine) denaturation. In the
presence of the probe, sc-dlFG was more susceptible
to denaturation by GdnHCl, pressure and heat.
Because its monomers are tethered to one another, we
believe that they maintain additional interactions
within the dimer, and that these interactions are affec-
ted negatively by the presence of the probe during
denaturation by guanidine, pressure and high tempera-
ture, and positively in the presence of urea. Although
the molecular basis of the effect of urea and GdnHCl
on polypeptide chains is still not well understood, it is
generally thought that urea mainly affects hydrogen
bonding. The binding of the probe presumably pro-
tects these interactions against urea.
Bis-ANS binding seems to be different from dlFG
and sc-dlFG, as the probe protected the dimer against-
most treatments and destabilized the fused form against
all treatments except urea-induced denaturation.
High hydrostatic pressure has been a very useful
tool in the study of folding intermediates, DNA recog-
nition, and virus assembly. Pressure studies have

revealed that there is a large step before total phage
assembly. The protein–DNA complex formed between
dlFG and poly(GC) DNA stabilizes the protein and
promotes the complete reversibility of the pressure
Fig. 8. Conformational changes in dlFG and sc-dlFG promoted by
high temperature. (A) The sample was treated at 25–80 °C, with an
incubation time of 5 min at each temperature. The structural chang-
es were analyzed by variation of spectral center of mass of trypto-
phan emission in the absence of bis-ANS for (m) dlFG, (r ) sc-dlFG.
and in the presence of bis-ANS for (e) sc-dlFG and the return at
room temperature for both in the absence and presence of bis-ANS
(d). The bis-ANS concentration was 1 l
M. (B) High-temperature-
induced denaturation of dlFG and sc-dlFG proteins analyzed by far-
UV CD spectroscopy. Conformational changes in (m) dlFG and (r)
sc-dlFG (in the absence of bis-ANS and (e) sc-dlFG in the presence
of 1 l
M bis-ANS) at increasing temperatures (15–85 °C). Ellipticity
values at 216 nm are plotted as a function of increasing tempera-
ture.
S. M. B. Lima et al. Bacteriophage MS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1471
denaturation process. We are unsure of the mechanism
of DNA-induced stabilization of the protein, and whe-
ther it is related to the normal RNA-binding function
of coat protein, as RNA binding had no effect.
The increased light scattering of samples after pres-
sure release in the presence of bis-ANS or DNA
suggests that the dimer is probably undergoing an
aggregation process. This suggests that, when the pro-

tein was submitted to high pressure, hydrophobic resi-
dues were exposed, allowing probe binding and
inhibiting the aggregation, but after pressure release
the probe bound to the protein may play a nucleation
role, inducing an aggregated state. A possible mechan-
ism is the formation of a limited associated state, for
example a pentameric unit, as observed for other ani-
mal viruses [29]. The binding of bis-ANS to the coat
protein dimer under pressure and the absence of pro-
tection from dissociation by DNA suggest that DNA
and bis-ANS may bind in the same or nearby sites of
the protein.
Genetically fusing the subunits of the dlFG dimer
greatly stabilized it against all the forms of denatura-
tion we tested. This is consistent with the observation
of increased stability of the single-chain version of the
wild-type dimer [30] and is reminiscent of the similar
stabilization engendered by subunit fusion in other
proteins [30–34]. This increased stability most likely
reflects the increased local concentration of one chain
with respect to the other when the two are covalently
tethered to one another.
Overall, our studies provide information on the
mechanisms by which the interactions in the capsid
lattice are made sufficiently stable and specific to allow
the formation of a correctly assembled particle, while
maintaining sufficient instability to allow release of the
viral genome during initiation of infection. Zlotnick
[35] discussed the importance of understanding virus
stability and assembly, pointing out that the virus must

assemble at the right time and in the right place so
as to package the correct nucleic acid. Moreover, it
must be able to undergo conformational transitions
to release its nucleic acid. The higher stability of the
capsid protein bound to heterologous nucleic acid may
serve as a ‘biological sieve’. Whereas authentic MS2
particles dissociate and unfold co-operatively, any
particle without the authentic RNA would be locked
in a state lacking the ability to release the RNA during
infection.
Our studies shed light on the processes that lead to
packaging of the correct RNA resulting in a mature
infectious viral particle. The efficient coupling of fold-
ing and assembly of the authentic virus particle
revealed by the co-operative processes of dissociation
and denaturation is very likely to play an important
role. The lack of this co-operativity for the VLP indi-
cates that, in the host, the complexes of capsid protein
bound to a small nonspecific RNA may lock it in the
disassembled state. There have been few studies that
addressed the topic of specific packaging of nucleic
acids by viruses. Annamalai et al. [36] described how
an arginine-rich RNA-binding motif, situated at the
N-proximal region of cowpea chlorotic mottle virus
(CCMV) capsid protein (CP), recognizes and packages
specific RNA. In the case of MS2 coat protein, it binds
to its cognate RNA hairpin 1000-fold tighter than the
corresponding DNA and any modification of one of
the 21 different RNA–protein contacts leads to a stri-
king change in the specificity of the RNA–capsid pro-

tein interaction [37]. It is the delicate balance between
proper protein–RNA affinity and thermodynamic sta-
bility of the resulting RNA packaged particle that
drives the formation of the infection virus.
Experimental procedures
Chemicals
All reagents were of analytical grade. Distilled water was
filtered and deionized through a Millipore water purifica-
tion system. The probe bis-ANS was purchased from
Molecular Probes (Eugene, OR, USA). The experiments
were performed at 20 °C using the standard buffer (10 mm
Tris ⁄ HCl, 100 mm NaCl, 0.01 mm EDTA, pH 7.5).
Phage propagation
E. coli cell strain C3000 was grown in Luria–Bertani med-
ium to A
600
¼ 1.2 when they were infected with MS2. After
5 h the culture was treated with lysozyme, and bacterial
debris was removed by centrifugation at 9800 g for 10 min
at 4 °C (RPR 9.2 rotor; Hitachi, Tokyo, Japan). The super-
natant was precipitated with ammonium sulfate (330 gÆL
)1
),
and the phage pellet was collected by centrifugation at
11 800 g for 45 min at 4 °C (RPR 12.2 rotor; Hitachi). The
precipitate was dissolved in standard buffer and purified by
high-speed centrifugation (155 000 g for 14 h; SW41 rotor;
Beckman, Fullerton, CA, USA) in a sucrose gradient (10–
50%). The phage was collected, and its purity determined
by SDS ⁄ PAGE (12.5% gel) and visualized by staining with

Coomassie Blue. Sample concentrations were determined by
Lowry’s method [38].
Expression and purification of VLPs
After growing overnight in Luria–Bertani medium, E. coli
cells strain BL21 (DE3) were transformed with a plasmid
Bacteriophage MS2 stability S. M. B. Lima et al.
1472 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
pETCT containing the sequence of wild-type coat protein.
The cells were diluted 20 times, and, after 2 h at 37 °C,
protein expression was induced with 1 mm isopropyl b-d-
thiogalactopyranoside. After 2 h the cells were pelleted by
centrifugation (5500 g for 20 min; RPR 9.2 rotor) at 4 °C.
The pellet was resuspended in standard buffer, treated with
lysozyme, sonicated and centrifuged (13 500 g for 20 min;
RPR 20.2 rotor; Beckman) at 4 °C. Cellular debris was
removed by centrifugation, and VLPs were then precipita-
ted with ammonium sulfate (330 gÆL
)1
) overnight followed
by centrifugation (13 500 g for 20 min) at 4 °C. Afterwards
the sample was applied to column of A-5M agarose (from
Bio-Rad). The coat protein was collected, and its purity
determined by SDS ⁄ PAGE (12.5% gel) stained with
Coomassie Blue. Sample concentrations were determined by
Lowry’s method [38].
Expression and purification of isolated dimer coat
protein (dlFG) and genetically fused coat protein
dimer (sc-dlFG)
The assembly-defective mutants dlFG and sc-dlFG were
propagated to mid-exponential phase (A

600
¼ 0.8) in strain
BL21(DE3) at 37 °C. Protein expression was induced with
2mm isopropyl b-d-thiogalactopyranoside. Three hours
after induction, the cells were centrifuged (5500 g for
20 min) at 4 °C and frozen at )20 °C overnight. After
being thawed, the cells were resuspended in lysis buffer
(0.02 m Na
2
HPO
4
, pH 7.4, 0.5 m NaCl, 0.001 m phenyl-
methanesulfonyl fluoride, 0.02 m 2-mercaptoethanol, 0.01 m
imidazole) and sonicated. The cell debris was pelleted by
centrifugation (13 500 g for 20 min). The supernatant was
added to Chelating Sepharose Fast Flow charged with
nickel ions and mixed gently for 30 min. The mixture
applied to a column, washed with increasing concentrations
of imidazole (20 mm,50mm, 100 mm, 250 mm), and the
protein was eluted with 500 mm imidazole. After purifica-
tion, the samples were dialyzed in buffer (0.01 m Tris ⁄ HCl,
pH 7.5, 0.1 m NaCl, 0.01 m EDTA).
Spectroscopic measurements and high pressure
experiments
Fluorescence spectra were recorded on an ISSK2 spectroflu-
orimeter (ISS Inc., Champaign, IL, USA). Tryptophan resi-
dues were excited at 280 nm, and emission was observed at
300–420 nm. Changes in fluorescence spectra were quanti-
tated by the spectral center of mass, <m>:
< m >¼ Rm

i
:Fi=RF
i
where F
i
is the fluorescence emitted at wavenumber m
i
. The
summation is carried out over the range of appreciable val-
ues of F.
For experiments in the presence of bis-ANS, the excita-
tion wavelength was 360 nm and emission was collected at
400–600 nm. For pressure experiments, the high-pressure
bomb has been described by Paladini & Weber [39] and
was purchased from ISS. The samples were allowed to
equilibrate for 20 min at each pressure before measure-
ments were recorded.
Light scattering
Light scattering measurements were made in an ISSK2
spetrofluorimeter. Scattered light was collected at an angle
of 90 ° to the incident light. The samples were excited at
320 nm and collected at the same wavelength. This wave-
length was chosen because neither protein nor RNA absorb
at 320 nm.
Chemical denaturation
The samples were incubated with increasing urea concentra-
tions (1–9 m) and increasing GdnHCl concentrations (0.5–
6 m) and allowed to equilibrate overnight before measure-
ments were recorded. The measurements were made in the
absence and presence of urea and GdnHCl.

CD
Conformational changes in MS2 bacteriophage, VLPs,
dlFG and sc-dlFG treated with urea and guanidine were
analyzed. The MS2 bacteriophage and the coat protein
mutant samples were diluted to a final concentration
100 lgÆmL
)1
, and the spectra were obtained in 10 mm
Tris ⁄ HCl ⁄ 30 mm NaCl, pH 7.5, using a 0.1-cm path length
quartz cuvette. The spectropolarimeter used was a Jasco
J-715 1505 model (wavelength range 300–210 nm).
Nucleic acid-binding assays
The effect of the presence of nucleic acids on the stability
of dlFG was analyzed during pressurization. The protein
was incubated with poly(GC) DNA, yeast tRNA and the
translational operator sequence of bacteriophage MS2
(5¢-AC AUGAGCA UUACCCA UGU-3¢) on r atio 1 : 2 ( nucleic
acid ⁄ protein). The sample concentration used was 2 lm, and
it was diluted in 0.01 mm Tris ⁄ HCl, pH 7.5, containing
0.1 m NaCl and 0.01 m EDTA.
Acknowledgements
We gratefully acknowledge Emerson Gonc¸ alves for
competent technical assistance, Cristiane Dinis Ano
Bom and Professors Fa
´
bio Almeida and Ana Paula
Valente from CNRMN ⁄ UFRJ for helpful comments
and suggestions. This work was supported in part by
an international grant from the International Centre
for Genetic Engineering and Biotechnology (ICGEB)

S. M. B. Lima et al. Bacteriophage MS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1473
to J.L.S. and by grants from Programa de Nu
´
cleos de
Excele
ˆ
ncia (PRONEX), Conselho Nacional de Desen-
volvimento Cientı
´
fico e Tecnolo
´
gico (CNPq), Fundac¸a
˜
o
de Amparo a
`
Pesquisa do Estado do Rio de Janeiro
(FAPERJ), Fundac¸a
˜
o Universita
´
ria Jose
´
Bonifa
´
cio
(FUJB) of Brazil to J.L.S. and A.C.O., and by a grant
from the National Institutes of Health (NIH) to
D.S.P.

References
1 Fiers W (1979) In Comprehensive Virology (Fraenkel-
Conrat H & Wagner RR, eds), pp. 69–204. Plenum,
New York.
2 Silva JL, Oliveira AC, Gomes AMO, Lima LMTR,
Mohana-Borges R, Pacheco ABF & Foguel D (2002)
Pressure induces folding intermediates that are crucial
for protein–DNA recognition and virus assembly.
Biochim Biophys Acta 1595, 250–265.
3 Ni CZ, Syed R, Kodandapani R, Wickersham J, Pea-
body DS & Ely KR (1995) Crystal structure of the MS2
coat protein dimer: implications for RNA binding and
virus assembly. Structure 3, 255–263.
4 Stockley PG, Stonehouse NJ, Walton C, Walters DA,
Medina G, Macedo MB, Hill HR, Goodman STS, Tal-
bot SJ, Tewary HK, et al. (1993) Molecular mechanism
of RNA-phage morphogenesis. Biochem Soc Trans 21,
627–633.
5 Lim F & Peabody DS (1994) Mutations that increase
the affinity of a translational repressor for RNA.
Nucleic Acids Res 22, 3748–3752.
6 Peabody DS (1993) The RNA binding site of bacterio-
phage MS2 coat protein. EMBO J 12, 595–600.
7 Romaniuk PJ, Lowary P, Wu HN, Stormo G & Uhlen-
beck OC (1987) RNA binding sites of R17 coat protein.
Biochemistry 26, 1563–1568.
8 Granh E, Stonehouse NJ, Adams CJ, Fridborg K,
Beigelman L, Matulic-Adamic J, Warriner SL, Stockley
PG & Liljas L (2000) Delection of a single hydrogen
bonding atom from the MS2 RNA operator leads to

dramatic rearrangements at the RNA–coat protein
interface. Nucleic Acids Res 28, 4611–4616.
9 Peabody DS & Lim F (1996) Complementation of
RNA binding site mutations in MS2 coat protein het-
erodimers. Nucleic Acids Res 24, 2352–2359.
10 Lima SMB, Peabody DS, Silva JL & Oliveira AC
(2004) Mutations in the hydrophobic core and in the
protein–RNA interface affect the packing and stability
of icosahedral viruses. Eur J Biochem 271, 135–145.
11 Pickett GG & Peabody DS (1993) Encapsidation of het-
erologous RNAs by bacteriophage MS2 coat protein.
Nucleic Acids Res 21, 4621–4626.
12 Axblom C, Tars K, Fridborg K, Orna L, Bandule M &
Liljas L (1998) Structure of phage fr capsids with a dele-
tion in the FG loop: implications for viral assembly.
Virology 249, 80–88.
13 Lago H, Parrott AM, Moss T, Stonehouse NJ & Stock-
ley PG (2001) Probing the kinetics of formation of the
Bacteriophage MS2 translational operator complex:
identification of a protein conformer unable to bind
RNA. J Mol Biol 305, 1131–1144.
14 Peabody DS & Ely KR (1992) Control of translational
repression by protein–protein interactions. Nucleic Acids
Res 20, 1649–1655.
15 Pushko P, Kozlovskaya I, Brede A, Stankevica E,
Ose V, Pumpens P & Grens E (1993) Analysis of
RNA phage fr coat protein assembly by insertion,
deletion and substitution mutagenesis. Protein Eng 6,
883–891.
16 Stonehouse NJ, Scott DJ, Fonseca S, Murray J, Adams

C, Clarke AR, Valegard K, Golmohammadi R, van den
Worm S, Liljas L et al. (1996) Molecular interactions in
the RNA bacteriophage MS2. Biochem Soc Trans 24,
412S.
17 Stonehouse NJ, Valegard K, Golmohammadi R, van
den Worm S, Walton C, Stokley PG & Liljas L (1996)
Crystal structures of MS2 capsids with mutations in the
subunit FG loop. J Mol Biol 256, 330–339.
18 Zlotnick A, Reddy VS, Dasgupta R, Schneemann A,
Ray WJ, JrRueckert RR & Johnson JE (1994) Capsid
assembly in a family of animal viruses primes an auto-
proteolytic maturation that depends on a single aspartic
acid residue. J Biol Chem 269, 13680–13684. 19.
19 Gray DM (1996) Circular dichroism of protein–nucleic
acid interactions. In Circular Dichroism and the Con-
formational Analysis of Biomolecules (Fasman GD, ed.),
pp. 469–500. Plenum Press, New York and London.
20 Weber G (1987) Dissociation of oligomeric proteins by
hydrostatic pressure. In High Pressure Chemistry and
Biochemistry. NATO-ASI Series (van Eldik R & Jonas
J, eds), pp. 401–420. D. Reidel Publishing Company,
Dordrecht.
21 Weber G & Drickamer HG (1983) The effects of high
pressure upon protein and other biomolecules. Q Rev
Biophys 16, 89–112.
22 Rosen CG & Weber G (1969) Dimer formation from
1-anilino-8-naphthalenesulfonate catalyzed by bovine
serum albumin. A new fluorescent molecule with
exceptional binding properties. Biochemistry 8, 3915–
3919.

23 Lim F, Spingola M & Peabody DS (1994) Altering the
RNA binding specificity of a translational repressor.
J Biol Chem 269 , 9006–9010.
24 van de Worm SHE, Stonehouse NJ, Valegard K, Mur-
ray JB, Walton C, Fridborg K, Stokley PG & Liljas L
(1998) Crystal structures of MS2 coat protein mutants
in complex with wild-type RNA operator fragments.
Nucleic Acids Res 26 , 1345–1351.
Bacteriophage MS2 stability S. M. B. Lima et al.
1474 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
25 Peabody DS & Chakerian A (1999) Asymmetric contri-
butions to RNA binding by Thr45 residues of the MS2
coat protein dimer. J Biol Chem 274, 25403–25410.
26 Da Poian AT, Oliveira AC, Gaspar LP, Silva JL &
Weber G (1993) Reversible pressure dissociation of R17
bacteriophage: the physical individuality of virus parti-
cles. J Mol Biol 31, 999–1008.
27 Silva JL, Foguel D & Royer CA (2001) Pressure pro-
vides new insights into protein folding, dynamics and
structure. Trends Biochem Sci 26, 612–618.
28 Silva JL, Foguel D, Da Poian AT & Prevelige PE Jr
(1996) The use of high hydrostatic pressure as a tool to
study viruses and other macromolecular assemblages.
Curr Opin Struct Biol 6, 151–156.
29 Oliveira AC, Ishimaru D, Gonc¸ alves RB, Smith TJ,
Mason P, Sa
´
-Carvalho D & Silva JL (1999) Low
temperature and pressure stability of picornaviruses:
implications for virus uncoating. Biophys J 76, 1270–

1279.
30 Peabody DS (1997) Subunit fusion confers tolerance to
peptide insertions in a virus coat protein. Arch Biochem
Biophys 347, 85–92.
31 Liang H, Sandberg WS & Terwilliger TC (1993) Genetic
fusion of subunits of a dimeric protein substantially
enhances its stability and rate of folding. Proc Natl
Acad Sci USA 90, 7010–7014.
32 Looker D, Abbott-Brown D, Cozart P, Durfee S, Hoff-
man S, Mathews AJ, Miller-Roehrkh J, Shoemaker S,
Trimble S, Fermi G, et al. (1992) A human recombinant
haemoglobin designed for use as a blood substitute.
Nature 356, 258–260.
33 White FL & Kenneth WO (1987) Effects of crosslinking
on the thermal stability of hemoglobin. The use of
bis-(3,5-dibromosalicyl)fumarate. Arch Biochem Biophys
258, 51–57.
34 Yang T & Kenneth WO (1991) Thermal stability of
hemoglobin crosslinked in the T-state by bis-(3,5-dibro-
mosalicyl)fumarate. Biochem Biophys Res Commun 174,
518–523.
35 Zlotnick A (2004) Viruses and the physics of soft
condensed matter. Proc Natl Acad Sci USA 101, 15549–
15550.
36 Annamalai P, Apte S, Wilkens S & Rao AL (2005)
Deletion of highly conserved arginine-rich RNA binding
motif in cowpea chlorotic mottle virus capsid protein
results in virion structural alterations and RNA packa-
ging constraints. J Virol 79, 3277–3288.
37 Dertinger D, Dale T & Uhlenbeck OC (2001) Modify-

ing the specificity of an RNA backbone contact. J Mol
Biol 314, 649–654.
38 Lowry OH, Rosenbrough NJ, Farr AL & Randall RJ
(1951) Protein measurement with the folin phenol
reagent. J Biol Chem 193, 265–275.
39 Paladini AA & Weber G (1981) Pressure-induced
reversible dissociation of enolase. Biochemistry 20,
2587–2593.
S. M. B. Lima et al. Bacteriophage MS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1475