Changes in the stability and biomechanics of 
P22 bacteriophage capsid during maturation
Authors: Ravi Kant, Aida Liauro, Vamseedhar Rayaprolu, 
Shefah Qazi, Pedro J. de Pablo, Trevor Douglas, & Brian 
Bothner
NOTICE: this is the author’s version of a work that was accepted for publication in Biochimica et 
Biophysica Acta. Changes resulting from the publishing process, such as peer review, editing, 
corections, structural formating, and other quality control mechanisms may not be reflected in 
this document. Changes may have been made to this work since it was submited for 
publication. A definitive version was subsequently published in Biochimica et Biophysica Acta, 
vol. 1862, issue 6, DOI#10.1016/j.bbagen.2018.03.006.
Kant, Ravi, Aida Llauro, Vamseedhar Rayaprolu, Shefah Qazi, Pedro J. de Pablo, Trevor 
Douglas, and Brian Bothner. "Changes in the stability and biomechanics of P22 bacteriophage 
capsid during maturation." BBA - Biochimica et Biophysica Acta 1862, no. 6 (June 2018): 
1492-1504. DOI:10.1016/j.bbagen.2018.03.006.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Changes in the stability and biomechanics of P22 bacteriophage capsid
during maturation
Ravi Kanta, Aida Llaurób, Vamseedhar Rayaprolud, Shefah Qazic,d, Pedro J. de Pablob,
Trevor Douglasc, Brian Bothnera,⁎
aDepartment of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, United States
bDepartamento de Física de la Materia Condensada and Solid Condensed Mater Institute IFIMAC, UAM, Francisco Tomás y Valiente 7, 28049 Madrid, Spain
cDepartment of Chemistry, Indiana University Bloomington, IN 47405, United States
dDepartment of Cel Biology and Neuroscience, Montana State University, Bozeman, MT 59717, United States
ABSTRACT
The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from
spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid ex-
pansion. Although P22 maturation has been investigated for decades, a unified model that incorporates ther-
modynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid ma-
turation is of significant interest to theoreticians searching for fundamental principles as wel as virologists and
material scientists seeking to alter maturation to their advantage. To address this chalenge, we have combined
the results from orthogonal biophysical techniques including differential scanningfluorimetry, atomic force
microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these
results from single particle and population measurements, an energy landscape of P22 maturation from pro-
capsid through expanded shel to wiffle bal emerged, highlighting the role of metastable structures and the
thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric ele-
ments of the capsid is a key component for stability in P22. A surprisingfinding is that the progression to wiffle
bal, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical
rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent
properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of
applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes
and provides a framework within which other viral systems can be compared.
1. Introduction
Supramolecular-protein complexes carry out many important biological
processes. These complexes require assembly, the directions for which are
programmed into the amino acid sequence and coordinatedviaalosteric
interactions. Icosahedral virus capsids are excelent systems for studying the
process of assembly. In viruses, assembly of a symmetric capsid is orche-
strated through a balance of polar and nonpolar interactions of asymmetric
subunits [1–3]. Assembly can often be divided into two steps, thefirst being
subunit association, folowed by an adoption of the active quaternary struc-
ture. The second step can involve covalent interactions and/or conforma-
tional changes [4–7]. In step two, subunit reorganization associated with
capsid maturation from spherical to quasi-equivalent icosahedral symmetry
can be classified into global and local rearrangements. Large global re-
organizations are for the most part irreversible and often involve the crossing
of a significant energy barrier on the conformational landscape. This is in-
itiated by a specific stimulus, such as, genome packaging, receptor binding, or
pH [4,8–10]. Local reorganizations are transient, resulting from the sampling
of different conformations, and have been referred to as capsid breathing
[11–13]. Beyond the obvious reasons for understanding viruses, there is in-
creasing interest in the use of protein-based nano-compartments in thefield
of applied nanotechnology [14–18]. These compartments are assembled from
multiple subunits and this self-assembly property can be exploited to in-
corporate different active molecules inside it. Viruses are pertinent examples
of precise, self-assembling, and highly stable nanoparticles. Knowledge and
understanding of a virus and its assembly and disassembly mechanisms can
be helpful to engineer a nanoparticle for a variety of bioinspired applications
[17–23]. The relationship between the assembly and disassembly pathways
depends on interaction between inter-protein subunits which further reg-
ulates the global stability and integrity of the capsid [24].
T
Among icosahedral viruses, P22 is a wel-studied model of assembly
and maturation. The capsid hasT= 7 icosahedral symmetry. During
assembly of the capsid and packaging of the genome, P22 undergoes a
series of structural transformations during which a spherical procapsid
adopts icosahedral symmetry (Fig. 1). This process of maturation results
in a 10% increase in diameter [25–27]. To assemble P22, 420 molecules
of coat proteins interact andco-polymerize with 60–300 molecules of
scaffold protein, 12 portal proteins, and 3 ejection proteins to form a
metastable procapsid [28–31]. Scaffold proteins are important both in
initiating assembly, and during polymerization, where they maintain
fidelity and active assembly [31]. The packaging of DNA is an ATP-
dependent process involving a terminase. During packaging, release of
electrostaticaly bound internal scaffold proteins is folowed by a re-
adjustment of the protein lattice and expansion of the capsid. Expansion
results in the formation of non-identical, quasi-equivalent contacts
between subunits. Quasi-equivalence demands a conformational
switching that involves the formation of hexamers and pentamers from
the same coat proteins [32]. Thus, smal differences in subunit inter-
actions and conformations must occur during P22 maturation. Al of
these changes are known to influence the free energy status of the ex-
panded form [26,33], but a connection between the biophysical
transformation and thermodynamics remains elusive.
The maturation process of P22 can be mimickedin vitroby applying
thermal or chemical stress. Heterologous expression of the coat and scaffold
protein results in self-assembly of the spherical procapsid (PC) with a dia-
meter of 58 nm. Heat or denaturants wil trigger expansion [26,28,34].
Heating of P22 procapsid at pH 7.6 mimics shel transformations that are
part of maturation (Fig. 1)[26]. Incubation of PC in 0.5 M guanidine hy-
drochloride (GdmHCl) releases scaffold protein, producing an empty shel
(ES) [35]. Heating of ES at 65 °C for 20 min irreversibly induces an ex-
panded shel (EX) with a diameter of 64 nm [36–38].HeatingofESat75°C
for 20 min causes the release of pentons fromfivefold axes producing the
wifflebal(WB)form[37,39]. Due to the ease of expression and the ability
to control maturationin-vitro, P22 has been established as a platform for
nanobiotechnology[16,18,22,40,41]. Thefirst three morphologies are
structural intermediates that are pertinent to the biology of P22 [37]. EX
and WB are currently being used as nano-protein containers for a wide
variety of cargos [42]. Change in pH is the natural trigger for initiating
maturation in a variety of viruses, including Cowpea chlorotic motle
viruses (CCMV) [43]andNudaureliaωcapensis [4]. Therefore, from a
nano-biotechnology perspective it is important to understand the effect of
pH on structural intermediates.
HK97 is a relevant model for capsid maturation. P22 and HK97 are
both bacteriophages and have a common subunit structure with highα-
helix content designated as the HK97 fold [44–46]. A comparative
study of structural intermediates of HK97 by atomic force microscopy
(AFM) showed an increase in Young's modulus and mechanical strength
after maturation [7]. The P22 maturation mechanism is different from
HK97 in that P22 does not form covalent cross-links, a unique feature of
HK97. Therefore, a comparison of these two capsids presents a unique
opportunity to investigate biophysical differences between covalent
and noncovalent protein complexes. Although numerous studies de-
scribe the global rearrangements associated with P22 maturation
[47,48], biophysical analyses are limited.
Biophysical and mechanical properties of proteins and protein
complexes can be investigated by differential scanningfluorescence
(DSF), circular dichroism (CD), atomic force microscopy (AFM) and
hydrogen deuterium exchange mass spectrometry (HDX-MS). DSF is an
established technique for folowing the thermal transitions of protein
complexes and viruses [49,50]. AFM can be used to image and make
measurements of deformation by mechanical force. It has been applied
to viruses such as HK97, PBCV, MVM and CCMV etc [51,52]. HDX-MS
is sensitive to changes in protein conformation and solvent exposure,
demonstrating great utility in the study of smaler and bigger protein
Fig. 1.Capsid forms and maturation of P22. (A)In vivomaturation event of Bacteriophage P22 which leads to infectious form. (B) It shows the different assembled
intermediates of P22 VLPs. Procapsid is composed of both coat and scaffold proteins, while Empty shel is devoid of scaffold proteins and only composed of coat
proteins. Expanded shel is enlarged icosahedral shel made up of coat protein. Wiffle bal is same as expanded shel except missing pentons fromfivefold vertices.
(PDB ID 3iyi-Procapsid, 2xyz-Expanded shel, Wiffle bal-3iyh).
complexes [6,53,54]. In the study presented here, we investigate the
biophysical mechanism behind P22 maturation across the four
morphologies (PC, ES, EX and WB). During maturation, when the
capsid accumulates genetic material, it undergoes a biophysical trans-
formation. Therefore, mature capsids need tofind a way to accom-
modate the change attributed to the presence of genetic material (in-
crease in size and pressure [54]) while maintaining structural integrity.
In thefield of physical virology, different studies have supported the
concept of coat protein-genome interactions [42,55,56] or inter-coat
proteins crosslinking for stabilizing a mature capsid [7].
By applying orthogonal techniques, we show that in P22, matura-
tion can be driven by tuning the weak quaternary interactions between
subunits, which results in a mature expanded capsid havingT=7
quasi-equivalent symmetry. As a result of this transformation, mature
P22 capsids are relatively rigid assemblies with a stabilized hydro-
phobic protein core and an enhanced global hydrogen bonding net-
work. We present a free energy model in which expansion is driven by
both enthalpic and entropic factors. This is in contrast with the model of
HK97 maturation, where rigidity is primarily established by covalent
crosslinking [7]. Ourfindings support a two-stage assembly in P22
maturation. In the first stage, assembly involves the formation of a
weaker structure (procapsid) with identical coat protein environment.
In the second step, the capsid expands resulting in a mature and stable
capsid with quasi-equivalence. These biophysical transformations also
result in an increased resistance to stresses such as heat and protein
denaturants. From the perspective of nanobiotechnology, we show that
just by altering the protein-protein interactions (PC/ES to EX) or re-
moving coat proteins interactions atfivefold axis (EX to WB), assembly
with altered biophysical and biomechanical properties is produced. The
orthogonal techniques presented here thus have broad implications in
thefield of virus biology and nano-biotechnology.
2. Materials and methods
2.1. Protein purification of WT P22
P22 WT procapsid made up of 420 subunits, was produced by a het-
erologous expression system inE.coli.BL21(DE)E.coliwas grown in 1 L
cultures inoculated with 1 mL starter culture (37 °C, 220 rpm). After 2 h
(OD600= 0.6), the cultures were induced with 1 mM IPTG and grown for 4
more hours. Cels were harvested by centrifugation at 3700gfor 20 min.
The cels were resuspended in PBS pH 7.6 and were incubated with DNase,
RNase, and lysozyme (al Sigma-Aldrich) for 30 min at room temperature.
Cels were lysed further by sonication on ice (Branson Sonifier 250,
Danbury, CT, power 4, duty cycle 50%, 3 × 5 min with 3 min intervals).
Bacterialceldebriswasremovedviacentrifugation at 12000gfor 45 min.
The supernatant was then loaded on a 35% sucrose cushion and centrifuged
at48,000rpmfor50mininanultra-centrifuge(50.2Tiultracentrifuge
rotor). The resulting virus pelet was resuspended in PBS, pH 7.0 and dia-
lyzed to remove sucrose. The P22 ES was prepared by repeated extraction
of the scaffolding protein with 0.5 M guanidine-HCl. Purified ES were he-
ated at 65 °C for 20 min to obtain P22 expanded and at 75 °C for 20 min to
obtain P22 wifflebal. Each individual capsid form was routinely char-
acterized using SDS-PAGE, native agarose gels, transmission electron mi-
croscopy (TEM, Leo 912 AB), and dynamic light scatering (DLS,
Brookhaven 90Plus, Brookhaven, NY).The protein concentration was de-
termined by absorbance at 280 nm using the extinction coefficient
(44,920 M−1cm−1).
2.2. Differential scanningfluorescence (DSF)
Differential scanningfluorescence scans of P22 morphologies (PC,
ES, EX and WB) were performed in citrate phosphate buffer pH 7
(prepared from stock, 100 mM citric acid and 200 mM disodium hy-
drogen phosphate). P22 samples were diluted in citrate phosphate
buffer with 50 mM NaCl concentration to obtain thefinal virus
concentration (0.15 mg/ml). To each sample, 2.5μL of 1% Sypro-
Orange dye (Invitrogen 140 Inc. S6651) was added to obtain thefinal
reaction volume to 25μL. The assays were conducted in a qPCR in-
strument (Corbett Research, RG-3000) with temperature elevating from
25 to 99 °C, increasing 0.5 degrees every 30 s. Lysozyme was run as
positive control atfinal concentration of 0.3 mg/ml. DSF assays were
also performed in different pH (2.6, 4, 5, 6, 8, and 9) citrate phosphate
buffer with the same experimental set up. Melting temperature of
samples was determined by calculating thefirst derivative (dF/dT)
from rawfluorescence data. For partial convoluted peaks, tip of the
peak is used to calculate the melting temperature. Thermal denatura-
tion of P22 samples were also studied in the presence offixed con-
centration of guanidine-hydrochloride concentration (0.5, 1, 2, 3 M) at
neutral pH. The reaction conditions and experimental set up in this
experiment were same as for different pH DSF assays.
2.3. Circular dichroic spectroscopy
Circular dichroic (CD) spectra were recorded on JASCO-815 spec-
trometer. CD measurements were performed on PC, ES, EX and WB
samples placed in fused silica cuvette with pathlength 10 mm (Starna
Cels, Atascadero, CA). Tightfitting stopper was placed on samplefiled
silica cuvette to prevent sample evaporation. CD spectra were per-
formed between 25 °C and 95 °C with the ramping rate of 1 °C/min
controled by a JASCO programmable Peltier-cooled temperature con-
troler. Final concentration of samples was maintained at 0.03 mg/ml.
For thermal dependent scan, the parameters were set in measure range
190–300 nm, scan speed of 100 nm/min, with a 1 nm bandwidth and
1 nm data pitch. The UV data was recorded at every 5 °C and the di-
chroic activity was supervised unceasingly and each observed spectrum
was an average of 3 scans.
2.4. AFM experiments
Measurements were performed with an AFM microscope (Nanotec
Electrónica S.L., Madrid, Spain) operating in Jumping mode plus [57].
Rectangular silicon-nitride cantilevers (Olympus, RC800PSA) with a
nominal spring constant of 0.05 N/m were used and calibrated using
Sader's method [58]. Maximum imaging force during the measurement
was 100 pN. The experiments were carried out under physiological
conditions. One 20μl drop of diluted stock solution was incubated on a
silanized glass surface [59] and, after 30 min, washed with buffer until
a volume of 60μl was reached. The tip was also pre-wetted with a 30μl
drop of buffer.
Al AFMimages wereprocessedusing WSxM software [60]. Capsids
stiffness and breaking force was determined as described in [61]. Viral
particles were considered as thin-shels that presented a linear elastic
response upon deformation with the AFM tip. The elastic constant
(stiffness) of the particle was obtained from the slope of the initial
linear part of the force-indentation curve. The breaking force was de-
fined as the maximum force reached during the deformation of the
particle, prior to the breakage of the shel. The critical strain was de-
fined as the ratio between the maximum indentation that the particle
withstands (corresponding to the deformation at the breaking force)
and the height of the particle (i.e., =εcritical δheightcritical, where is the criticalindentation).
2.5. Hydrogen deuterium exchange mass spectrometry (HDX-MS)
Conformationalflexibility of P22 samples were studied by intact
protein hydrogen deuterium exchange mass spectrometry. This ex-
periment was carried out on purified PC, ES, EX and WB capsids se-
parately. Exchange reaction was instigated by 10 fold dilution of P22
stock concentration by D2Obuffer (10 mM NH4HCO3,50 mM NaCl,
pH 7.4). Final concentration of al samples after dilution was 0.1 mg/
ml. Hydrogen deuterium experiments were performed on 1290 UPLC
series coupled to micro-TOF spectrometer (Bruker Daltonics) operated
in positive electrospray ionization mode. Auto sampler was set to draw
samples from the vial at different time points (0.5, 2.5, 5.5. 12.5, 37.5,
60.5, 90.5, 720 min). For Quenching of reaction, HxSil C8 reversed
phase Hamilton column (20 × 2.3 mm), frit porosity 2μm is pre-equi-
librated with acid quenching conditions [20% (v/v) acetonitrile, 80%
H2O and 0.1% formic acid (w/v), pH 2.4]. The gradient conditions were
set as folows: 0.5 min, 40%B; 0.5–0.7 min, 40–95%B; 1.3 min, 95%B,
1.5 min 20% where solvent A: 0.1% formic acid in water and solvent B:
0.1% FA in acetonitrile. Theflow rate was 0.5 ml/min, auto sampler
and column compartment temperature were set at temperature 25 °C
and 37 °C. Data processing and analysis was performed with the help of
Bruker Data Analysis package version 4.0. The maximum entropy de-
convolution algorithm was used to calculate the increase in the mass of
the P22 sample at different time points in the spectral range of 800m/z
to 1600m/z, which covers the majority of subunit charge envelope.
Mass of deuterated samples at different points is calculated and the
mass of control sample is subtracted from them to determine the total
exchanged deuteriums. The relative mass difference between deuter-
ated and undeuterated samples were calculated and plotted against
time.
2.6. Calculation of solvent excluded surface area
The cryo-EM structures of P22 were not solved at atomic resolution
[49,62] and could not provide the positions of the side chains. Side-
chains were necessary to compute the solvent excluded surface areas of
subunits. To address this issue, Cαchains from the cryo-EM structures
(2xyy, 2xyz, 3iyi, 3iyh) were extracted and were submitted to the
Phyre2 server [63] for a template based side chain structure modeling.
In brief, the P22 amino acid sequence was submitted to the Phyre2
server [63] along with Cαchain extracted from the cryo-EM structure.
The Cαchain was used as the template to make sure the output model
structuraly aligns wel with the cryo-EM. During this process, the side-
chains were also predicted and included in the output model. Parent
et al…solved the structures 3iyi and 3iyh whereas Chenet alsolved the
structures 2xyy and 2xyz. In the case of 3iyi and 3iyh, the Cαstructures
were missing a number of residues (~40 amino acids). This discrepancy
was accounted for during sidechain modeling by submitting only the
sequence seen in the structure. To account for quasi-symmetry and for
conformational polymorphism, Cαchains from multiple subunits were
submitted to the Phyre2 server. For each of the structures obtained from
Phyre2, clashes were eliminated and refinement was done using Mod-
Refiner [64]. The refined models were then structuraly aligned into
their respective quasi and icosahedral positions in the P22 capsid using
the cryo-EM structure as the guide. Solvent excluded surface area
(SESA) was computed from these aligned models with a probe radius of
~1.4 Å for each subunit using UCSF Chimera software [65]. The
structure alignment was done using the matchmaker tool in UCSF
Chimera software and SESA calculations were also performed in the
same software. A guide cage was created to correctly position the
modeled protein subunits on their respective icosahedral axes. As the
procapsid has a more spherical structure, the sphere factor of the guide
cage (29 nm radius) created was set to maximum. Similarly, sphere
factor for the expanded shel and the wiffle bal guide structures (34 nm
radius) was set to the minimum to show the icosahedral nature of the
actual capsid (Fig. 2.7 A, B). Each corresponding subunit in al the
forms iscolored thesame.The numeric values for specific interactions
are listed in the table in the supplemental material (Table S1).
3. Results and discussion
3.1. Differential scanningfluorescence reveals heat triggered structural
transitions
P22 maturation involves a significant expansion of the capsid driven
by subunit rearrangement [62]. This prompted us to test if changes in
particle stability were associated with the transitions. In order to
measure the thermal stability of P22 bacteriophage pre and post ex-
pansion, DSF was used. DSF is a high throughput, straightforward, and
sensitive technique for tracking thermal unfolding transitions of pro-
teins. It uses afluorescent dye that is quenched in an aqueous en-
vironment. An increase influorescence intensity is observed upon in-
teraction with buried hydrophobic regions of a protein [66,67]. This
technique is useful for studying thermal transitions and measuring the
thermal stability of viruses and virus-like particles [50]. DSF thermal
scans of PC, ES, EX and WB were performed from 25 to 99 °C at pH 7.0.
The scan of PC, which contains both coat and scaffold proteins, ex-
hibited three transitions labeled I, I, II at 43.5 °C, 75 °C, and 88.5 °C,
respectively (Fig. 2,Table 1). Heating of ES, which no longer contains
scaffold protein, had two thermal transitions that matched I and II
from PC. The EX form of capsid also displayed two transitions, again
matching I and II. WB, which is missing the pentamers, showed a
single transition at the same temperature as II.
A comparison of DSF scans of PC and ES revealed the absence of
transition I in the ES (Fig. 2). The major difference between PC and ES
is the absence of scaffold, strongly suggesting that transition I is asso-
ciated with scaffold release. Galisteo and King demonstrated that there
is no distinct thermal transition during unfolding of scaffold proteins
possibly due to a general lack of tertiary structure [26]. Transition I was
broad and occurred at relatively low temperature (30 °C to 50 °C),
which further supports our interpretation. A comparison of thermal
profiles from PC, ES, EX and WB revealed the absence of transition I in
PC
ES
EX
WB
I I II
Fig. 2.DSF analysis of P22 capsids. PC, ES, EX and WB were heated from 25 to
99 °C (pH 7.0) while monitoringfluorescence intensity of Sypro Orange.
Thermal scan of PC (top) shows the presence of three transitions (I, I, II) which
correspond to different stages of maturation (as shown inFig. 1). ES, which has
lost scaffold proteins shows only two transitions (I, II). EX also shows two
transitions (I, II), while WB, which is devoid of pentamers has only one
transition (II).
WB. WB is structuraly similar to EX, as both have icosahedral sym-
metry and a diameter of 64 nm. The difference is the absence of pen-
tamers in WB. Together, this data strongly advocates that transition I is
the release of pentamers. However, transition I is relatively broad,
suggesting a non-cooperative process. Therefore, release of pentamers
would occur independently at each of the 12 5-fold vertices.
Previous experiments using differential scanning calorimetry (DSC)
to analyze P22 maturation observed three endothermic transitions [26].
The temperature of these transitions (48.5 °C, 71.4 °C and 87.3 °C)
correspond with what we have observed by DSF. Apart from the above
three transitions, a single exothermic transition was also observed by
DSC at 61 °C.Electronmicroscopy, size exclusion chromatography and
agarose gel electrophoresis also confirmed the release of scaffold at
48.5 °C and expansion of procapsid at 61 °C. Complete denaturation of
particles was reported at 85 °C [26]. This data is consistent with our
DSF experiments. However, only three transitions were observed by
DSF compared to the four transitions in DSC. The transition related to
expansion was absent, suggesting that once PC has lost scaffold, the
hydrophobic core of the capsid has largely been established and re-
folding involving exposure of hydrophobic regions does not occur. It is
only after ES has transitioned through EX and the pentamers are re-
leased to form WB that the second DSF transition is observed.
Structural studies indicate that large movements of pentons along
thefivefold axes are similar both inin-vivoandin-vitroexpansion [68].
Difference maps ofin-vivoandin-vitroexpanded particles show note-
worthy similarity, further validating the use of thermaly induced
transition to study the maturation of P22 [37]. The release of pentamers
is not anin-vivoevent, rather it is only observed during heat-induced
maturation. This loss of pentons suggests a destabilization of penton
and hexon units at higher temperature and a possible mechanism for
relieving stress in an icosahedral particle. Continuum elasticity theory
predicts that there is high lateral compression in pentamer units and
that this stress increases significantly with Triangulation number [69].
Previous work on human adenovirus mechanics has experimentaly
demonstrated that pentons are under stress [70]. A previous in-
vestigation has demonstrated that the penton moves radialy outward
by ~37 Å upon heating. This movement is folowed by the release of
pentamers at the icosahedralfivefold axis [68].In-vivo, the interaction
between capsid protein and the DNA genome has been proposed to
stabilize the pentons, averting release of pentamers during expansion
and DNA packaging [37]. These transitions are independent, and we
propose thateach formofcapsid occupies a local energy minimum.
Transition I is associated with the release of scaffold protein, transition
I is associated with the release of pentamers at thefivefold axis, and
transition II is thefinal denaturation of expanded capsids.
3.2. Impact of pH on thermal transitions
While temperature is a convenient tool for nudging capsids over
energy barriers associated with maturation, it is of course not a part of
the biological process. A more relevant parameter is pH, which can
initiate maturation by altering electrostatic interactions between
subunit interfaces. The smal icosahedral RNA tetravirus,Nudaureliaω
capensis(NωV), can be induced to mature from procapsid to capsid by
decreasing the pH from 7.0 to 5.0. The mature capsid form is mor-
phologicaly distinguishable, and smaler in diameter [4,71]. Another
example is cowpea chlorotic mottle virus (CCMV), which at pH 5.0 is
found as a closed 28 nm particle. At pH 7.0 and low ionic strength,
CCMV transitions to a swolen fenestrated form with the diameter in-
creasing by 10% [72]. Inspired by these studies, we were interested in
the impact of pH on heat induced structural transitions in P22. To test
this, DSF of each form was repeated at pH 2.6, 4.0, 5.0, 6.0, 8.0 and 9.0.
DSF results at pH 5.0–9.0 showed the same transitions and similar
temperatures as those observed at pH 7.0 (Fig. 3). At pH 4.0 PC tran-
sition I was lower by 5 °C, transition I by 3 °C, and transition II by 4 °C
(Table 1). No significant changes in Tmwere observed for transitions
associated with ES, EX and WB at pH 4.0. This observation for PC
suggests that at low pH, the interaction of scaffold and coat protein is
disrupted and scaffold protein is released relatively earlier. This early
release of scaffold results in partial destabilization of the coat protein
interactions in the PC, thereafter al the successive transitions occurred
at relatively lower temperature (Fig. 3). P22 particles remain intact at
even lower pH, however, DSF scans at pH 2.6 revealed that they were
destabilized. The DSF scan of PC at pH 2.6 had only two transitions, the
first (I/I) centered at 37 °C and second transition (II) centered at
62.5 °C. ES and EX had two transitions (I and II) at slightly higher
temperatures than in PC (Table 1). As expected, WB showed only the
final transition (II) at 65.5 °C, associated with particle disruption.
These results show that with the exception of the PC, al forms of P22
capsid remained stable between pH 4–9. Below pH 4, P22 particles
showed substantial thermal destabilization. But the relative intensity of
transition II increased in the order of PC < ES < EX/WB suggesting
high thermal stability after expansion (Table 1). From this, we conclude
that from a biological perspective pH has little effect on P22, however,
in use as a nanomaterial, disassembly of cargo bound capsid can be
triggered by dropping the pH to 2.5. But this low pH may influence the
cargo biophysical characteristics, therefore, pH below 4 should be
considered with caution.
3.3. Maturation increases chemical resistance
To gain a deeper understanding of the nature of capsid protein in-
teractions during maturation, impact of the chemical denaturant
GdmHCl was investigated. At low concentration, 0.5 M, GdmHCl is used
to release scaffold protein without changing the integrity of capsid
[35]. At higher concentrations, GdmHCl alters the free energy of
folding, making the folded form less favorable. Studies of model pro-
teins indicate that the ionic nature of GdmHCl masks electrostatic in-
teractions of proteins leading to unfolding [73–75]. A recent study in-
volving solvent effects on protein unfolding has also reported the
destabilizing effect of GdmHCl on proteins by facilitating the aqueous
solvation of non-polar side chains and back bone surface area [76].
Irrespective of the mechanism, GdmHCl is a commonly used tool for
investigating protein denaturation.
Table 1
Transition temperature from DSF analysis of P22 morphologies at different pHs.
P22 samples Temp range (°C) Transitions pH 2.5 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9
PC 30–45 I 37.0 ± 0.5a 38.0 ± 0.5 42.0 ± 16 43.5 ± 16 43.5 ± 16 43.5 ± 16 43.5 ± 16
70–80 I 37.0 ± 0.5a 72.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16
80–90 II 62.5 ± 0.1 84.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5
ES 70–80 I 39.0 ± 0.5 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16
80–90 II 63.5 ± 0.1 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5
EX 70–80 I 39.5 ± 0.5 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16 75.0 ± 0.16 88.5 ± 0.5
80–90 II 64.5 ± 0.1 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5
WB 80–90 II 64.5 ± 0.1 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5 88.5 ± 0.5
aTm of transition I and I is same for procapsid at pH 2.6 as these events are convoluted.
1496
DSF thermal scans of PC, ES, EX and WB were performed at different
concentrations of GdmHCl (0.5, 1, 2 and 3 M) at pH 7.0. In 0.5 M
GdmHCl, PC Showed a decrease in intensity of transition I and partial
convolution of transitions I and II (Fig. 4A). Compared with untreated
particles, temperature of transitions I and II were lowered by 2 °C and
6 °C respectively (seeTable 2). GdmHCl release of scaffold from pro-
capsid could account for the near absence of transition I. (Fig. 4A). This
interpretation is consistent with a previous study where incubation in
0.5 M GdmHCl was shown to remove scaffold subunits from purified
procapsids. The DSF curve of ES revealed a partial convolution of
transition I & II. This partial convolution of transitions I and II
suggests they are less distinct energeticaly. In ES thefluorescence in-
tensity ratio of transitions I:II was nearly 1:1, whereas in PC, transition
I intensity was higher than transition II (Fig. 4A). The melting tem-
perature of ES transitions I and II were centered at 73 °C and 82.5 °C as
wel as for PC (Table 2). For EX, there is a reduction in the intensity of
transition I, this could be due to the release of a smal subset of pentons
prior to transition.
Heating of PC at a higher concentration (1 M GdmHCl) resulted in
the complete convolution of transitions I and II into a single transition.
PC ES
EX WB
2.6
4.0
5.0
6.0
7.0
8.0
9.0
2.6
4.0
5.0
6.0
7.0
8.0
9.0
A) B)
C) D)
2.6
4.0
5.0
6.0
7.0
8.0
9.0
2.6
4.0
5.0
6.0
7.0
8.0
9.0
Fig. 3.Effect of pH on thermal stability of P22. DSF analysis of PC, ES, EX & WB from pH 2.6 to 9.0. A) DSF scans of PC show the presence of three transitions (I, I,
II), except at pH 2.6 Panels B-D show data for ES, EX, and WB, respectively. For each capsid form, only thermal scans conducted at pH 2.6 differed significantly from
the others. P22 thermal transition are independent of pH from 4 to 9. Plots show normalized intensity of three independent experiments. Values on the right of each
plot indicate pH.
PC
PC
ES
EX
WB
0M
0.5M
0.5M
0.5M
0.5M
0M
1M
1M
1M
1M
A) B) Fig. 4.DSF monitoring of chemical stabilityof P22. PC, ES, EX & WB analyses was per-
formed in increasing concentrations of gua-
nidine hydrochloride. A) 0.5 M Gdm-HCl. B)
1 M Gdm-HCl. Plots are the normalized
average intensity of three independent ex-
periments. Higher concentrations of Gdm-
HCl are show in supplemental material.
This suggests the concomitant release of pentamers and particle dena-
turation. ES behaves much the same, with the exception of a very smal
shoulder peak in the latter half of the transition (Fig. 4B), indicating
that it maintains a structural transition to a greater extent than a PC.
The melting temperature of the convoluted transitions for both PC and
ES was centered at 68.5 °C (Table 2). In contrast to the PC and ES, EX
retained transitions I and II distinctly. The melting temperature of the
prominent transition II was 84 °C for both EX and WB. In 1 M GdmHCl
thermal transitions differed by nearly 11 °C between non-expanded (PC,
ES) and expanded particles (EX, WB) for transition II. Heating in 2 M
GdmHCl resulted in significant loss of structural transitions for PC and
ES, contrary to EX and WB which retained distinct transitions (Table 2
and Fig. S3A).
Heating in 3 M GdmHCl showed complete loss of al transitions for
each morphology (Fig. S3). The addition of GdmHCl had a more pro-
nounced destabilization effect on spherical (PC and ES) compared to
icosahedral capsids (EX and WB). This data is consistent with study on
another Icosahedral T7 capsid where mature capsid exhibited resistance
to GdmHCl mediated global denaturation [77]. In DSF, melting tem-
perature is defined as the temperature at which maximal binding of the
hydrophobic dye SYPRO Orange is observed. Therefore, DSF reports on
the destabilization of hydrophobic protein regions. Our denaturant
study confirms that once the P22 particle matures, the hydrophobic
core is stabilized and can better resist denaturant. This observation is
supported by Raman spectroscopy of P22, PC and EX which revealed
that al six tryptophans in PC readily exchange hydrogens, whereas
after conversion to EX exchange decreases [47]. The large increase in
Tm associated with expansion suggests that the changes in protein
stability go beyond the tryptophan residues.
3.4. Protein subunit refolding does not have a significant role in maturation
To gain further insight into the structural changes associated with
P22 maturation, we turned to Circular Dichroism (CD), which provides
information specific to protein secondary structure. It is useful for the
study of local or global protein unfolding as a function of temperature
or denaturants [78–80]. Spectra of al four P22 morphologies were
recorded from 25 to 95 °C. Our CD data showed that P22 capsid sec-
ondary structure is rich inβstrands. This data is consistent with pre-
vious CD data colected on purified coat proteins [33] and structural
model [62]. Analysis of the thermal scans showed no significant change
Table 2
Transition temperature from DSF analysis of P22 samples.
P22 samples Transitions 0.5M GdmHCl 1M GdmHCl 2M GdmHCl
PC I 34.5 ± 0.5 – –
I 73 ± 0.5 – –
II 82.5 ± 0.5 68.5 ± 0.16 53.5 ± 0.16
ES I 73 ± 0.5 – –
II 82.5 ± 0.5 69.5 ± 0.16 56 ± 0.16
EX I 73 ± 0.5 70 ± 0.16 56.5 ± 0.16
II 84 ± 0.5 81.5 ± 0.16 77.5 ± 0.16
WB II 84 ± 0.5 82 ± 0.16 78 ± 0.16
Fig. 5.AFM analysis of P22 morphology. (A) AFM
images of individual P22 particles before (left) and
after the breakage (right). From top to bottom:
procapsid, empty shel (lacking scaffolding pro-
tein), expanded capsids (along the 5-fold symmetry
axes) and wiffle bal (along the 3-fold symmetry
axes). Color bar, from substrate to the highest
point: grey-red-yelow-white. (B) Force-indentation
curves corresponding to the particles shown in
panel A. (C) Comparison of the height, (D) elastic
constant (k), and (E) critical strain (εcritical) for the
different morphologies (mean ± SE). For EX and
WB the symmetry could be distinguished and par-
ticles were classified in two groups (S5 and S3/S2).
in the secondary structure from 25 to 85 °C at pH 7.0 (Fig. S1). How-
ever, above 85 °C there was a sharp transition in the CD curves, fol-
lowed by a complete loss of signal (Fig. S1). The spectra do not tran-
sition through a random coil signal, but rather a loss of signal. This
suggests that subunit dissociation was folowed by rapid aggregation
and precipitation. The temperature transitions observed by CD, DSF,
and DSC are highly similar [26]. Our CD experiments argue against
secondary structural changes as a driving force for the global structural
transitions.
3.5. AFM analysis shows an increase in mechanical rigidity with maturation
Our data showed that maturation of P22 increases particle stability
to heat and denaturant. This led us to ask whether this translates into
changes in mechanical properties as wel. To investigate this, we
complemented our biochemical experiments with single particle mea-
surements by AFM. This analysis was conducted in liquid, alowing us
to identify and characterize the structure and height of the different
particles under experimentaly relevant conditions (Fig. 5A and B). PC
and ES particles were spherical with heights of 50 ± 1 nm and
48 ± 1 nm, respectively (black and red bars,Fig. 5B andTable 3).
These values correspond to 86% and 83% of particle diameter as re-
ported by cryo-EM [62], suggesting that capsids were deformed by
contact with the surface. This observation is consistent with another
study on P22 VLPs [24]. The EX particles presented a more faceted
appearance and their orientation could be resolved (for example, the EX
capsids shown inFig. 5A were adsorbed along the 5-fold symmetry axis
(S5)). EX particles presenting a S5 orientation were higher than parti-
cles sitting on the 2- or 3-fold axes. This trend disappeared for WB
particles, for which the three orientations presented similar heights
(Fig. 5B).
Stiffness and brittleness are important material properties and can
be measured by nano-indentation. This entails deforming individual
particles with an AFM tip until the capsid breaks [52]. The deformation
of the particle (indentation) is registered as a function of the force
applied, which leads to the force-indentation curve (FIC) [82].Fig. 5C
shows four examples of FICs. These FICs correspond to the deformation
and breakage of the particles shown in panel 5A (left and right, parti-
cles before and after breakage). From each FIC, a value of stiffness
(elastic constant, k) and brittleness (critical strain,εcritical) can be ob-
tained [83]. High values ofεcriticalindicate that the particle can with-
stand large deformations without rupturing, whereas low values are
characteristic of brittle materials. Similarly, high values of stiffness
indicate that more force is required to cause deformation.
No significant changes in the mechanical properties were observed
after scaffold protein removal (black and red bars,Fig. 5D and E).
However, after expansion, particles increased in rigidity and brittleness
(blue bars,Fig. 5D and E). WB particles were less rigid, as predicted by
continuum elasticity theory, [33] and their capsids were less brittle.
3.6. Hydrogen-deuterium exchange of P22 capsids
Each of the biophysical analyses performed showed a consistent
increase in capsid stability up to the EX form. We hypothesized that
changes in the hydrogen bonding network within and between protein
subunits were responsible. This could explain the increased stability of
the hydrophobic core as wel. To test this, hydrogen deuterium ex-
change mass spectrometry (HDX-MS) was used. HDX-MS is a straight-
forward and versatile technique for probing the stability of protein
hydrogen bonding and dynamics of protein complexes [6,53,54]. P22
particles were rapidly diluted 10-fold in a buffered solution of D2O. The
incorporation of deuterium was measured over time using LCMS [84],
with the rate and extent of deuterium uptake inversely correlating with
hydrogen bond stability. The P22 coat protein had a measured mass of
46,620 Da (expected 46,620). After dilution in D2O, a rapid uptake of
deuterium was observed for al four morphologies during thefirst 5 min
of exchange (Fig. 6A). EX showed the least uptake in comparison with
others. The 0–5-min time window represents fast exchanging regions.
Fast exchange is generaly attributed to solvent exposed and dynamic
domains. These are often coils, loops, and regions undergoing con-
formational change that lack stable hydrogen bonding [85,86]. After
one hour of exchange, PC and WB had a significantly greater level of
exchange than ES and EX. The later time points report on medium to
slow exchanging amide protons, providing information on protein re-
gions with greater hydrogen bond stability. The reaction was folowed
for 12 h to ensure that slow exchanging regions, those with the most
stable hydrogen bonding networks, were accounted for. Slow ex-
changing regions report on wel-ordered secondary, tertiary, and qua-
ternary structures. At 12 h, samples had a progressive decrease in ex-
change from PC > ES > EX, with 18 additional residues per subunit
protected on average (Fig. 6B). The observed decrease in exchange is in
agreement with our hypothesis that maturation leads to a more stable
hydrogen bonding network across the capsid. However, WB showed an
increase in exchange compared to EX. This observation was initialy
unexpected due to the fact that WB showed a higher Tmand resistance
to chemical denaturation. We believe this is due to the absence of
Table 3
AFM measurements of P22 particles.
Capsids # of particles K εcritical Height
N/m nm
PC 32 0.094 ± 0,006 0.18 ± 0.01 50 ± 1
ES 12 0.105 ± 0,005 0.17 ± 0.01 48 ± 1
EX S5 8 0.22 ± 0.02 0.12 ± 0.01 62 ± 1
WB S5 3 0.17 ± 0.04 0.26 ± 0.05 52 ± 6
EX S3/S2 11 0.20 ± 0.02 0.14 ± 0.02 56 ± 1
WB S3/S2 9 0.13 ± 0.01 0.16 ± 0.03 51 ± 1
Fig. 6.Intactproteinhydrogendeuterium
exchange mass spectrometry of P22 particles.
PC, ES, EX & WB were diluted in 10 mM
ammonium bicarbonate, 50 mM NaCl D20
buffer, pH 7.0. (A) Number of deuterium in-
corporated (y-axis) along with incubation
time (x-axis) for coat protein. (B) Total
number of deuteriums exchanged after 12 h.
Total exchange reactions were carried out for
12 h and done in three technical replicates.
Error bars represent ± SD.
pentamers, which increases the average solvent accessible surface area,
making direct comparisons between WB and other forms difficult to
interpret by HDX-MS.
3.7. Maturation increases solvent excluded area
If our hypothesis that a wel-established hydrophobic core and a
more robust hydrogen bonding network is ultimately responsible for
the increased stability of P22 capsids as they mature, there should be
evidence for this in the structural models. Models of P22 from cryo-EM
at 4.0 Å resolution showed that the change in the morphology from PC
to EX involved a change from skewed to symmetric hexamers [62].
Symmetric hexamers create a consistent interaction surface across
hexameric and pentameric capsomers and alow additional interactions
in the E loop (G60-A67) and P domain (G396-S402) as wel [62]. This is
suggestive of increased global interactions in the mature capsid. How-
ever, a direct analysis of subunit interfaces and solvent excluded sur-
faces had not been completed. To do this, we analyzed changes in
quaternary organization, paying specific attention to the total solvent
excluded surface area (SESA) of each P22 form. Two sets of cryo-EM
based structural models from two different labs were used for this
analysis. Due to the requirement for side chain density to accurately
estimate SESA, the cryo_EM models (PDBfiles 2xyy, 2xyz [62] 3iyi,
3iyh [39]) were submitted to Phyre2 [63] and Modrefiner [64] for side
chain modeling and secondary structure refinement.
The two data sets represented different morphological pairs corre-
sponding to PC and EX [62] and PC and WB [64]. We compared the
refined models for EX and WB with their respective PC partner (2xyy
with 2xyz and 3iyi with 3iyh). Our goal was to make global compar-
isons that took into account the quaternary structural components and
would match closely to the global biophysical data above. This ap-
proach would also de-emphasize differences specific to the respective
pairs of models.Fig. 7A and B show the subunit groups from different
cryo-EM models used in the SESA calculations. Panel C shows the re-
lative difference in SESA values at regular (I) and quasi-equivalent (Q)
axes for the two pairs (See Fig. S2 for details). The numerical values of
SESA (Å2) were obtained for different subunits and percent change is
calculated by subtracting SESA (Å2)unexpanded from expanded form
and divided by total SESA (Å2) (Supplementary Table S1,Fig. 7C).
Percent positive change shows higher SESA after maturation, that fur-
ther indicates higher order of inter-coat protein interactions. Com-
parative analysis of PC: 2xyy and EX: 2xyz showed an increase in SESA
at both regular icosahedral and quasi-equivalent axis: I3 (2.2%), I5
(7.7%), Q3 (5.0%), Q5 (7.7%) and Q6 (2.4%). The comparison of PC:
3iyi and WB: 3iyh showed I3 (12.0%), Q3 (1%), Q5 (20%) and Q6
(8.8%). However, the comparison of PC: 3iyi and WB: 3iyh showed a
decrease in SESA at Q-3 and I-5. This was expected because WB lacks
pentamers, so residues facing the I-5 axis are more solvent accessible.
This is consistent with our HDX on WB, where an increase in exchange
was observed (Fig. 6). Despite lacking pentamers, WB shows a higher
SESA in al other axes. This confirms that each subunit in the mature
form contributes more to the central hydrophobic core than in its
procapsid counterpart. Our experimental observations are supported by
a computational study on multiple icosahedral viruses (deposited in
VIPER databases), where they have proposed that virus assembly may
be driven by burying hydrophobic surface area at inter-protein surfaces
[87,88]. A study involving computational analysis of quaternary in-
teractions in icosahedral viruses demonstrated that the association en-
ergy is an indicator of stabilities at quasi-equivalent interfaces [89]. We
extracted the association energy from VIPER [90] and compared across
both models (PC:2xyy and EX:2xyz, PC:3iyi and WB:3iyh) at common
interfaces. There is a significant increase in calculated association en-
ergy of EX and WB, the two forms that are quasi-equivalent (Table S2A,
S2B). Inspecting our SESA calculations along with the AFM results, we
suggest that an increase in intra-protein hydrophobic interactions due
to capsid expansion lead to a more rigid and brittle EX particle. After
removal of the 5-fold subunits, a substantial loss in terms of SESA, the
WB particle has a comparable rigidity but significantly lower brittle-
ness. In WB, intra-protein interactions are concentrated around the
icosahedral and quasi-icosahedral 3-fold and 5-fold axes. Interestingly,
in both SESA comparisons, we observed an increase in the solvent ac-
cessibility at I2 and Q2 axes. A previous study on human Adenovirus
found that the two fold axis is the stiffest in the capsid [91]. The SESA
calculations are consistent with the idea that mechanical anisotropy
arising from structural differentiation at axes of symmetry contributes
to the capsid stiffness [92]. During the preparation of this manuscript,
an atomic resolution cryoEM structure of the P22 mature capsid (PDB:
5UU5) was released [48]. Calculations revealed that the new EX
structure had greater SESA due to the inclusion of more electron density
in thefinal model. However, the overal trend remained the same.
3.8. Maturation stabilizes capsids by enhancing quaternary interaction
The maturation of P22 capsids has been under investigation for
several decades, however, the spatial arrangement of coat proteins en
route to maturation that leads to the specific biophysical attributes in
the context of thermodynamics hasn't been discussed. Since the ma-
turation event is irreversible and thermodynamics tels us only about
states, we have discussed the PC, ES, EX and WB as metastable forms
occupying separate energy wels. These wels are separated by an ac-
tivation energy/kinetic barrier which can be overcome by specific
temperature and/or chemical triggers. Therefore, we combined the data
colected here to produce an energy landscape model. (Fig. 8). Positions
lower on the plot have lower free energy (greater thermodynamic sta-
bility). A series of local energy minima are shown as shalow wels, the
width of which reflects the conformational freedom as measured for
each form. Broad energy wels represent structures with relatively
greaterflexibility, while a narrow energy wel represents a more rigid
structure. Based on our results, the conformational ensemble re-
presenting PC and ES are shown as broad energy wels. This is sup-
ported bytheir greatersolventaccessibility (measured by HDX-MS) and
lower rigidity (measured by AFM). The DSF and HDX data show ES to
be more stable than PC. Therefore, ES occupies a lower position than PC
on the free energy axis (Fig. 8). Our results indicate EX is more stable
than ES, thus it is lower again onFig. 8. The narrow energy wel for EX
reflects the higher stability, rigidity, brittleness and relative increase in
SESA as revealed by DSF, HDX, AFM and structural models, respec-
tively.
An important question remains about the driving force mediating
the transition from spherical to icosahedral form. Our intact protein
HDX-MS indicated a net increase in hydrogen bond network stability, a
finding that was not included in previous HDX studies that focused on
exchange in specific regions [93,94]. This suggests an enthalpic con-
tribution to maturation which is supported by an earlier DSC study
which found a negative change in enthalpy was associated with ex-
pansion [26]. Calculation of SESA from structural models showed a
relative increase after maturation indicating an increased hydrophobic
contribution to the shel after maturation. When hydrophobic regions
are buried and excluded from solvent, it results in the release of bound
water with a net increase in the system entropy at the expense of a local
reduction in entropy. Therefore, we now suggest that the change in P22
morphology from PC to EX (maturation) is mediated by both enthalpy
and entropic driven factors.
The WB which has lost its pentamers, eliminating protein-protein
interactions atfive-fold axes, remarkably remains resistant to heat and
denaturant. But it is less rigid and exchanges more deuterium than EX
(Figs. 5 & 6). Limited proteolysis of WB shows that release of pentamers
exposes a cleavage site to Trypsin [39], analogous to the increased
exchange. The increased deformability could be indicative of con-
formational entropy which balances the lost inter-subunit interactions.
WB is the most stable form of P22 to heat and chemical stress, which
may seem surprising given the net loss of subunit contact area, but in
the context of icosahedra, pentamers represent defects in the hexagonal
lattice. The existence of WB elegantly demonstrates the complexity
inherent in capsid protein interactions and the emergent properties that
can arise from icosahedral symmetry.
Finaly, we can summarize that the assembly and maturation pro-
gram for P22 is encoded in the amino acid sequence of the coat protein
and is regulated by a two-step mechanism. Thefirst step in assembly is
initiated using relatively weak interactions between coat subunits and
scaffold proteins leading to a spherical procapsid (PC). Weak interac-
tions are a critical requirement for the precise positioning of coat pro-
teins to avert the formation of aberrant structures and kinetic traps.
Hence, the procapsid represents a higher energy local minimumen-
routeto the more stable expanded forms. The second step in the process
is dictated by quaternary interactions and rules of quasi-equivalence
(EX/WB) which require subunits to inhabit non-identical local en-
vironments. This step is achieved through conformational switching
once expansion is initiated.
4. Conclusion
The thermodynamics and molecular mechanisms driving assembly
and maturation of icosahedral virus capsids are complex. Bacteriophage
capsids are faced with a difficult chalenge in that they must increase
stability as they expand. In a cel, this is triggered by DNA packaging
and in many cases, requires protein-DNA interactions to gain stability.
Through the use of orthogonal analytical techniques involving single
particle and population based methods, we have now worked out a
model that can be used to discuss general and specific features of ex-
pansion mediated icosahedral capsid maturation. P22 manages this
thermodynamic balancing act without the aid of covalent cross-linking,
which is used by HK97. The general biomechanical transformations
described here for P22 provide a model for other viral systems which
including dsDNA viruses such as adeno and herpes. Thesefindings can
also guide design programs for the development of icosahedral capsids
as nanomaterials and containers where tunable stability and rigidity are
of significant value.
Transparency document
TheTransparency documentassociated with this article can be
found, in the online version.
Fig. 7.Solvent excluded surface area (SESA)
of P22 capsids. (A) Spacingfiling models of
selected subunits show the transformation
between PC: 2xyy and EX: 2xyz 54 (B)
Spacingfiling models of PC: 3iyi and WB:
3iyh38display the transition from PC to WB.
Absence of pentamers is observed around
fivefold axis in WB. Solvent excluded surface
area (SESA) was computed with a probe ra-
dius of ~1.4 Å using UCSF Chimera software
103after the addition of side chain density.
(C) Percent difference in SESA is calculated
and compared across both pairs (2xyy and
2xyz, black color column bar) and (3iyi and
3iyh, grey column bar) along with regular
icosahedral axes (I) at 2, 3 and 5 fold and
quasi-equivalent (Q) at 2, 3, 5 and 6 fold
axes. Numeric SESA values (Å2) is calculated
for each axis by averaging al the interacting
subunit SESA values at a given axis (Table
S2). Asterisk * at I-5 indicates a reduction in
SESA for WB.
Acknowledgements
The authors would like to thank Jonathan K. Hilmer for technical
support with Mass Spectrometry and Liam Scott for critical reading of
the manuscript. The Proteomics, Metabolomics, and Mass Spectrometry
Facility at MSU has received support from the Murdock Charitable Trust
and National Institute of General Medical Sciences of the National
Institutes of Health under Award Number P20GM103474. This work
was made possible in part by funding to BB from the National Institutes
of Health NIAID AI081961-01A1. PJ. P and A. Ll. Acknowledge projects
FIS2014-59562-R, FIS2015-71108-REDT, Fundación BBVA and“María
de Maeztu”Program for Units of Excelence in R&D (MDM-2014-0377).
Appendix A. Supplementary data
Supplementary data to this article can be found online athttps://
doi.org/10.1016/j.bbagen.2018.03.006.
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