SURFACE CHARGE DENSITY EFFECT ON
HBV CAPSID ASSEMBLY BEHAVIOR IN SOLUTION
A Thesis
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Xinyu Sun
May. 2016
SURFACE CHARGE DENSITY EFFECT ON
HBV CAPSID ASSEMBLY BEHAVIOR IN SOLUTION
Xinyu Sun
Thesis
Approved:
Accepted:
________________________________
Advisor
Dr. Tianbo Liu
_____________________________
Dean of the College
Dr. Eric Amis
________________________________
Co-Advisor
Dr. Toshikazu Miyoshi
________________________________
Dean of the Graduate School
Dr. Chand Midha
________________________________
Department Chair
Dr. Coleen Pugh
________________________________
Date
ii
ABSTRACT
Large, hydrophilic inorganic ions like polyoxometalate (POM) clusters were found
able to self-assemble into single-layered, hollow, spherical “blackberries”, and long range
electrostatic interaction was believed to be the major driving force.1 During the
blackberry formation study, a sigmoidal kinetic curve was observed when macroions
assemble with the existence of high salt concentration, indicating a two-step process. 2
Interestingly, blackberry structure and formation process share many similarities
with virus capsids. A large amount of virus capsids, e.g. Hepatitis B virus (HBV), have
singled-layered spherical structures and also show a process of nucleation when being
assembled by protein subunits with the addition of monovalent or divalent salt in vitro.3,4
Capsid formation has long believed to be dominated by hydrophobic interaction and the
role of electrostatic interaction was not much discussed in the capsid formation process
although the protein subunits are polyelectrolytes and can also be regarded as soluble
macroanions. So in our study, two charged amino acids on capsid protein are mutated
into neutral ones, and the effect of charge density on capsid structure and formation
tendency were proposed.
iii
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my adviser, Dr. Tianbo Liu, for
leading me into this interesting area, and providing me with the opportunity to give a shot
on this project. I also want to address my great appreciation to Prof. Yinan Wei at
University of Kentuchy for her group’s great effort in providing samples for us.
Besides I would like to thank to Dr. Toshikazu Miyoshi for being my committee
member and giving me some new ideas and helpful suggestions, and also Dr. Dong Li
and Dr. Panchao Yin for their introduction and great help on experiments. Finally, I
appreciate all my group members in Dr. Tianbo Liu’s group for their help and inspiration,
and my families for supporting me in everything.
iv
TABLE OF CONTENTS
Page
LIST OF FIGURES.......................................................................................................... vii
LIST OF TABLES..............................................................................................................ix
CHAPTER
I. BACKGROUND AND INTRODUCTION .................................................................... 1
1.1 Introduction of hepatitis B virus (HBV) and its capsid ........................................ 1
1.2 HBV capsid formation .......................................................................................... 3
1.3 Solution behavior of macroions ............................................................................ 7
1.4 Study motivation ..................................................................................................11
II. EXPERIMENTAL SECTION ...................................................................................... 13
2.1 Sample Design and Preparation .......................................................................... 13
2.2 UV-Visible .......................................................................................................... 14
2.3 Laser Light Scattering (LLS) .............................................................................. 14
2.4 Transmission Electron Microscope ..................................................................... 14
2.5 Sucrose density gradient ..................................................................................... 15
III. RESULTS AND DISCUSSION.................................................................................. 16
3.1 Capsid formation monitored by Dynamic Light Scattering (DLS) .................... 16
3.2 Capsid structure analyzed by sucrose gradient and TEM ................................... 20
3.3 Assembly tendency difference between WT and D2ND4N dimers ................... 23
IV. CONCLUSION ........................................................................................................... 26
BIBLIOGRAPHY.............................................................................................................. 27
v
LIST OF FIGURES
Figure
Page
1. A cartoon illustrating HBV virus composition ............................................................... 2
2. Three-dimensional maps of hepatitis core particles........................................................ 2
3. Truncations to capsid protein for assembly test and its corresponding proportion of
T=4 capsids ......................................................................................................................... 4
4. Cp149 assembly study in different pH conditions .......................................................... 4
5. Induction of 5 μM Cp149 assembly by different kinds of cations.................................. 5
6. Different sodium chloride concentrations used to induce the assembly of 5 μM Cp149
dimer solutions in 0.1 M sodium bicarbonate (pH 9.5) buffer at 20°C .............................. 6
7. Schematic plot of blackberry structure formed by POM{Mo154}................................. 8
8. Comparison of scattered intensity increment of two {Mo72Fe30} samples .................. 9
9. Model of Cp149 dimer with mutated or important sites for intra-dimer and inter-dimer
interactions ........................................................................................................................ 12
10. Scattered intensity increment of WT and Mutant dimers triggered by salt ................ 17
11. CONTIN analysis of DLS study on WT and D2ND4N dimer with continuous addition
of salt ................................................................................................................................. 18
12. DLS results of WT capsid solution at different angles ............................................... 19
13. Capsid size of WT and mutant vs. time ...................................................................... 19
14. Sucrose density gradient analysis of assembled Cp149 WT and D2ND4N by KCl in
buffer N ............................................................................................................................. 21
15. Negative staining TEM images for WT and mutant capsids from fractions after
sucrose density gradient ultracentrifuge ........................................................................... 22
16. WT and D2ND4N capsid formation tendency comparison ........................................ 24
vi
LIST OF TABLES
Table
Page
1. Polyoxometalate (POM) Molecular Clusters .................................................................. 7
vii
CHAPTER I
BACKGROUND AND INTRODUCTION
1.1 Introduction of hepatitis B virus (HBV) and its capsid
Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular
carcinoma in humans and has been chronically infecting millions of people. HBV still
catches people’s attention because despite the invention of many antiviral drugs, which
can effectively disturb virus assembly or replication, they still cannot save those who
have already infected.5-7
HBV belongs to the HepadnaViridae family and is an enveloped DNA virus with an
icosahedral core. This core, the capsid, is protected by surface proteins outside and is
composed of the viral nucleic acid, reverse transcriptase, and the protein capsid (figure
1).8,9 The structures of the capsid have been characterized to be icosahedral symmetries,
with a majority of T=4 icosahedral symmetry and part of T=3 structure, consisting of 240
and180 subunits respectively.9,10 Figure 2 gives three-dimensional maps of both T=3 and
T=4 hepatitis core particles, achieved by image reconstruction, in both exterior and
interior views.
1
Figure 1: A cartoon illustrating HBV virus composition. Copyright:
http://www.ibibiobase.com/projects/hepatitis/hepatitis-aB.htm
Figure 2: Three-dimensional maps of hepatitis core particles. (a) and (c):Exterior and
interior views for T=4 icosahedral particles; (b) and (d): Exterior and interior views for
T=3 icosahedral particles. Copyright: 1996 American Chemical Society.
2
1.2 HBV capsid formation
The assembly and disassembly of virus capsid play an important role in the virus life
cycle, since the assembled protein coat is stable enough to protect the nucleic acid
genome, while the disassembly with response to certain change in environment can
release the genome for replication. Virus capsid formation is of much complexity but
always shows high fidelity. In vitro, the nucleocapsid protein HBcAg with and without
the existence of genome both have the ability to assemble into capsids, and these capsids
showed no difference in morphology and antigenic properties.3,11 Core protein dimer is
believed to be the basic building block for capsid assembly since no monomer was
detected in experiments. The protein hydrophobic domain helps its dimerization and the
intrinsic disulfide bond stabilizes the dimer structure. 12
Among 183 amino acids of HBcAg polypeptide, the last 34 residues in C-terminal
region (residues 150-183) are rich in positively charged arginines, whose role is thought
to be binding DNA inside the capsid and the rest proteins participate in building the
capsid structure.13 Interestingly, this assumption was proved by a series of experiments
performed in Dr. Zlotnick’s group by truncating HBcAg into shorter capsid proteins (Cp).
Cp149 dimer is the capsid protein with the last 34 amino acids removed, and it can still
assemble into capsid when dialyzed against 0.1 M HEPES buffer with 0.2-0.5 M NaCl.
Capsid proteins can maintain their capsid assembly ability even if their lengths are
shorten into Cp147, Cp142 and Cp140, however, larger extent of truncation in Cterminus will decrease T=4/T=3 capsid ratio until Cp138 can no longer form any
capsid.10 In these studies, high concentration of extra salt was a necessary and it was
3
assumed to induce the conformational change in the protein, so that an assembly-active
conformation can be adopted for the assembly 14.
Figure 3: Truncations to capsid protein for assembly test and its corresponding proportion
of T=4 capsids. Copyright 1996 American Chemical Society.
Figure 4: (A) During dialysis of dimeric Cp149 (0.5 mg/ml) in pH 9.5 buffer against 50
mM Tris-HCl (pH 7.0) buffer with 250 mM NaCl for 12-14 h at 4 oC, its assembly
dependency on pH was revealed by determining dimer and capsid concentration through
sedimentation velocity. (B) The salt dependency was studied in pH 7.0 buffer with
corresponding amount of NaCl at protein concentration of 0.5 mg/ml. Copyright 1995
American Chemical Society.
4
More detailed exploration into Cp149 assembly revealed that HBV assembly is a
function of protein concentration, ionic strength and solution pH. The dimer assembly is
much favored at lower pH (e.g. pH 7.0 or 7.5) and higher salt concentration (Figure 4 A,
B). In pH 7.0 solution, 250mM NaCl is enough to trigger 0.5 mg/ml protein assembly,
while most proteins keep dimer status at this protein and salt condition in pH 9.5 buffer. 3
Except for sodium chloride as ‘initiator’ for the capsid assembly induction, other monoand divalent cations were also used to exam the influence of ionic strength. For 5 μM
Cp149 in 50 mM HEPES pH 7.5, as illustrated in figure 5, all buffered salts were added
into dimer solution and then incubated for 16 h at 21 °C. Na+ and K+ ions played a
function in assembly when their concentrations reached 0.2 M, while 50 mM of Mg2+
and Ca2+ ions were enough. In contrast, only 200 μM of Zn2+ and Ni2+ could trigger an
obvious assembly, suggesting a selective and specific binding. 15
Figure 5: Induction of 5 μM Cp149 assembly by different kinds of cations. Experiments
were performed in 50 mM HEPES buffer at room temperature and the assembly was
monitored by 90° light scattering. Copyright 2004 American Chemical Society.
5
HBV capsid assembly at pH 9.5 is less favored, thus providing chance to do its
kinetic study. In this situation, at least 0.5 M NaCl is required to induce 1 mg/ml protein
dimer assembly. So at low salt concentration or protein concentration, a lag phase
indicating the kinetically hindered period can be observed (Figure 6). But this lag phase
will be shortened when salt or protein concentration is increased. The extent of capsid
assembly shows a third-order concentration dependence while the assembly rate exhibits
a second-order concentration dependence based on simulation result, which means the
assembly is initiated by formation of a trimeric nucleus and then followed by the
accumulation of these nucleuses.4
Figure 6: Different sodium chloride concentrations used to induce the assembly of 5 μM
Cp149 dimer solutions in 0.1 M sodium bicarbonate (pH 9.5) buffer at 20°C. The
increment of scattered intensity was recorded by a fluorometer at 320 nm. Copyright
1999 American Chemical Society.
6
1.3 Solution behavior of macroions
Normally, small ions smaller than 1 nm like NaCl can form real solution following
Debye-Hückel theory, while those larger than that (1~1000 nm) will form insoluble
suspension and balanced by van de Waals attraction and repulsive electrostatic
interaction as described by Derja-guin-Landau-Verwey-Overbeek theory (DLVO
theory).16-18 But the page was blank on macroions when these soluble ions reach the size
of nanometer scale and can no longer be treated as point charges until our group devoted
much to this field.19
Table 1. Polyoxometalate (POM) Molecular Clusters
The giant hydrophilic polyoxometalates (POMs, listed in table 1) are taken as simple
models to represent large inorganic molecules18. POMs carry inherent charges or partially
deprotonated water ligands, with counter-ions surrounded, working as the major driving
force to their stable aggregation state in dilution solution. Dynamic light scattering (DLS)
gives angular independent sizes at different angles and static light scattering (SLS) gives
a radius of gyration (Rg) close to hydrodynamic radius (Rh), indicating the aggregation
7
has a hollow spherical structure. This new type of self-assembled structure was given a
nickname called ‘blackberry’, and figure 7 illustrates the blackberry structure.20
Charge density on POMs can be tuned by changing the solution pH or the dielectric
constant of mixture solvent. In a certain charge density range, when macroions carry less
negative charges, stronger association with counter-ions will decrease the blackberry
curvature and increase the blackberry size.21-22 Since macroion size is kept no change,
van der Waals force cannot be the major contributor to its interesting solution behavior.
Figure 7: Schematic plot of blackberry structure formed by POM{Mo154}. A single layer
of POMs are evenly distributed on the blackberry surface and its hollow spherical
structure was confirmed by DLS, SLS and Zimm plot. Copyright 2003 Nature Publishing
Group.
8
Extra salt like K+ or Rb+ can screen electrostatic interaction and adjust attractive
force, leading to reversible change in blackberry size.23 Except for this, salt can also
kinetically slow down the formation process and bring a significant lag phase at the
beginning of the macrion self-assembly (figure 8). The lag phase refers to the slow
formation of oligomer nucleus; once the amount of the rate-limiting nucleus has reached
a critical value, subsequent oligomers or monomers are quickly added to the growing
assembly structures at a time until it is complete 2.
Figure 8: Comparison of scattered intensity increment of two {Mo72Fe30} samples: full
circle represents {Mo72Fe30}with 0.9 wt % NaCl; empty square refers to salt-free
solutions. Copyright 2009 American Chemical Society.
Besides, POM macroions exhibit a lot of wisdom in selecting the right partners
during the self-assembly. For example, {Mo72Cr30} and {Mo72Fe30} can both form into
blackberries independently with different radiuses. When mixed together, however, they
can still recognize the oligomers of their own type and grow into blackberries of two
9
sizes, each equal to that formed in pure solution of their own 24. Chiral recognition of two
enantiomeric wheel-shaped macroanions was also reported, along with the achievement
of chiral separation, which means chiral co-anions are able to selectively suppress the
aggregation of one of the enantiomers.25
All these studies show that POM macroions self-assembly is a controllable selective
process, sharing some similarities with bio macromolecules in building virus capsid.
More resemblance can be found when comparing capsid structure and assembly process
of blackberry and virus: both are hollow spheres (icosahedron for most virus); a
sigmoidal kinetic curve with a lag phase can be observed for both systems; and ionic
strength is playing a regulation role. 2,19
Although some differences can be found between these two, like relative stable
capsid size with a fixed number of subunits v.s. tunable blackberry size responding to
ionic strength change; and extra counter-ion can shorten lag phase for capsid formation
but do the opposite for blackberry, our curiosity is aroused to explore more influence of
electrostatic interaction in building virus capsid. 2
10
1.4 Study motivation
Many studies have been done to investigate the key determinants in capsid
formation, including changing or deleting several amino acids on capsid dimer. Figure 9
shows three important sites on which mutation study had been conducted. Dimer-dimer
contact sites is a popular study area because any interface change may favor or disfavor
assembly and change capsid stability. The dimer-dimer interface contact is dominated by
residues 125 to 142 at either end of the dimer,26 so mutation had been performed on
amino acid R127 and Y132 respectively. R127L, R127Q and Y132A, going through
either change in interface charge or hydrophobicity, failed to assembled into capsid,
indicating the importance of dimer-dimer interface.27,28 Another study of interest lies in
Cys61, two of which will oxidize into an intradimer disulfide bond after the capsid is
formed, helping stabilizing the capsid.29 However, if Cys61 was replaced with Ala,
dimerization and assembly were not influenced with the absence of the disulfide, except
for more T=3 capsids were found 10.
Bio-molecules are also polyelectrolytes and belong to macroions, so it is reasonable
to assume the capsid protein assembly behavior may be influenced by regulating its
charge. So far no research has specifically worked on the effect of charge density on
capsid formation. Thus it would be exciting to compare capsid growth with blackberry
and explore how much difference can electrostatic interaction contribute to.
11
Figure 9: Model of Cp149 dimer with mutated or important sites for intra-dimer and
inter-dimer interactions marked in yellow. Cp149 dimer is composed of two Cp149
subunits shown in blue and red respectively. Amino acid 97 in yellow is a naturally
occurring mutation site. Residue Cys61 in yellow is involved in the intra-dimer disulfide
bond formation. Residues 125 to 142 in yellow are important sites for dimer-dimer
contact. The original model was downloaded from RCSB RDB. 30
12
CHAPTER II
EXPERIMENTAL SECTION
2.1 Sample Design and Preparation
Isoelectric point of wild-type (WT) is 4.71, thus marking it negatively charged in
pH>7 solution. To study the influence of charge, two negatively charged Aspartic acids
(D) are mutated into neutral amino Asparagines (N) on each subunit and its assembly
process is compared with WT protein. The mutation sites are carefully chosen to avoid
those involved in salt bridge. Finally, D2ND4N dimer, with four charges less than WT
dimer, was chosen as the study target. The assembly process was studied in buffer N (50
mM NaHCO3, 10 mM BME, pH=9.6) at room temperature since proteins should carry
more charges and the charge effect can be more obvious. AT pH 9.6, WT dimer has -12.8
*2 charges, while D2ND4N dimer has -10.8 *2, calculated from protein calculator 3.4
(http://protcalc.sourceforge.net). Both samples are nicely provided by Prof. Yinan Wei
from University of Kentuchy.
13
2.2 UV-Visible
UV-visible spectra were measured by HEWLWTT PACKARD G1103A UV-Vis
spectrometer at room temperature. UV absorbance at 280 nm was recorded and then
calculated into protein concentration with an extinction coefficient of 29280 cm-1M-1 and
path length of 1 cm.
2.3 Laser Light Scattering (LLS)
Dynamic light scattering (Brookhaven Instrument LLS spectrometer, laser
wavelength 532nm) was used to monitor the protein assembly process and to determine
the hydrodynamic radius Rh. Intensity-intensity time correlation can be measured by DLS
first and then convert into field correlation function, which will be further analyzed by
CONTIN method.31 The normalized distribution function of the characteristic linewidth
G(Γ) can be used to determine the average diffusion coefficient, D=Γ/q2. The
hydrodynamic radius Rh can be obtained from diffusion coefficient through the StokesEinstein equation: Rh = kBT/(6πηD), in which kB is the Boltzmann constantand η the
viscosity of the solvent at temperature T. All the DLS data were collected at room
temperature.
2.4 Transmission Electron Microscope
To visualize the capsids, negative staining TEM was operated by depositing 8 uL of
solution containing core particles on a carbon-coated copper grid for 1 min, then slightly
14
suck off the extra solution and wash the grid with droplets of water and stain with 8 uL of
1% uranyl acetate for 1 min. Transmission electron micrographs of negatively stained
capsids were taken by JEOL-1230 microscope.
2.5 Sucrose density gradient
To further analyze the capsids, sucrose density gradient was adopted to differentiate
assembled core particles. 0.3 mL of capsid solution was loaded onto 10% to 40% filtered
sucrose solution buffered with buffer N and 1M K+ till a total volume of 5 mL. Both
samples were spined in a Ti-55 rotor (performed by Beckman ultracentrifuge) at 45,000
rpm at a temperature of 20oC for 2 h. The resulted solution was separated into 10
fractions and then taken out from top to bottom. UV absorbance at 280 nm was measured
for each faction and the absorbance was normalized according to the first fraction.
15
CHAPTER III
RESULTS AND DISCUSSION
3.1 Capsid formation monitored by Dynamic Light Scattering (DLS)
Both WT and mutant dimer can be stable in stock solution buffer N until the assembly
was triggered by salt. The capsid formation can be monitored by DLS because capsids are
larger than dimers and they can scatter more light, thus bringing increase in scattered
light intensity. Lower movement of capsids can differentiate themselves from dimers and
present a hydrodynamic radius of capsids.
In our study, potassium chloride was chosen to promote Cp149 assembly. At the
beginning, pure dimer solution showed low intensity since dimers just have an Rh around
3 nm. As soon as a high concentration of buffered salt was added into dimer solution, an
increase in the scattered light intensity was observed, indicating the dimers were
aggregating into oligomers and adding up together to form larger capsids. Figure 10 is a
plot of scattered intensity increment vs. time recorded at room temperature. Normally the
assembly process is finished within several days. Once the intensity comes stable, it
means assembly equilibrium has been reached, and capsid size is also kept stable. During
this period of time, the dimer peak at ~3 nm decreases and a new capsid peak (~17 nm)
16
appears and continues to grow, as shown in figure 11a and figure 11b. DLS
measurements took at different scattering angles showed no angular dependence of the
capsids, indicating a spherical morphology of the capsid (figure 12).
Figure 10: Scattered intensity increment shows the assembly of WT and D2ND4N
triggered by salt (1.301 mg/mL) WT with 1.08 M K+ and 1.478 mg/mL D2ND4N with
1.136 M K+ accordingly). The relative light scatter value was the result of (Isample – I buffer
N)
/ IBenzene .
17
a
b
Figure 11: CONTIN analysis of DLS study on WT (a) and D2ND4N (b) dimer with
continuous addition of salt. Peak of Rh lower than 10 nm is corresponding to dimer, while
the larger one is capsid.
18
Figure 12: DLS results of WT capsid solution at different angles.
Figure 13: Capsid size of WT and mutant vs. time
Based on previous studies, WT capsid has an overall diameter of 36 nm for T= 4
particles and 32 nm for T=3 measured to the spikes tips. These spikes are formed by fourhelix bundles of the dimer and have an approximate length of 25 Å and width of 20 Å.
Therefore, if only considering the shell size, it is 15 nm in radius for T=4 structure and 13
19
nm for T=3.9,32 From our DLS study, WT capsids have an average radius of 17.3±0.5 nm
and D2ND4N capsid 11.7±1.0 nm (figure 13). WT capsid size is accord with literature
results measured form electron cryomicroscopy and X-ray crystallography, but mutant
D2ND4N always shows a smaller average capsid size. This size difference could due to
(1) It requires more dimer proteins to form one WT capsid than that does the mutant
capsid; (2) The dimer-dimer distance in the mutant capsid is closer and smaller than that
of WT capsid; (3) Large oligomers exist in mutant solution that may shift the capsid peak
to a smaller size.
3.2 Capsid structure analyzed by sucrose gradient and TEM
To further investigate the capsid size difference, both WT and D2ND4N capsids were
analyzed by sucrose density gradient. After ultracentrifuged in 10% to 40% sucrose
solution for 2 hours, the resulted solution separated into 10 fractions were taken for UV
absorbance test, and the existence of Cp149 core protein or capsid was determinated by
the UV absorbance at 280 nm. Figure 14 normalized UV absorbance results according to
the first fraction. Because of the low molecular weight of the dimer proteins, they stayed
in top fractions. Meanwhile, WT capsids dominate fraction No. 6 (TEM image in Fig 5a)
and No. 8 (TEM image in Fig 5b), while D2ND4N capsids showed smaller sediment
ability with their appearance at fraction No. 7 (TEM image in Fig 5c). Fraction No.5 of
the mutant solution has a high UV absorbance but capsids were not seen under TEM, so
this UV absorbance may mainly come from protein oligomers. According to TEM, WT
capsids have an average diameter of 26 nm and 30 nm for fraction No. 6 and No. 8
20
accordingly, exactly corresponding to T=3 and T=4 structure. D2ND4N capsids in
fraction No. 7 have an average size of 30 nm in diameter.
D2ND4N capsids in fraction No. 7 exhibited smaller sediment coefficient than WT
fraction No. 8 but they almost have the same size under TEM. This sediment ability
difference can hardly come from change in molecular weight caused by mutation, since
D2ND4N just has 2 less Dalton on each protein compared to WT Cp149, so capsid MW
difference is 480 Dalton in maximum (if considering 240 subunits on each capsid), which
is neglectable compared to capsid molecular weight (4 million Dalton). So if there is any
structural difference (e.g. dimer-dimer distance or number of dimers on each capsid)
between these two waits explored by cryo-EM and 3D reconstruction analysis.
Figure 14: Sucrose density gradient analysis of assembled Cp149 WT and D2ND4N by
KCl in buffer N. Resulted solutions were separated into 10 fractions from top to bottom
and UV absorbance of each fraction was recorded and normalized according to the first
21
fraction. Plot in the right is a zoomed-in sketch, illustrating capsids with different
sediment coefficients.
Figure 15: Negative staining TEM images for WT and mutant capsids from fractions
after sucrose density gradient ultracentrifuge: (a) WT capsids from sucrose fraction
No. 6; diameter 26 nm in average; (b) WT capsids from sucrose fraction No. 8;
diameter 30 nm in average; (c) Mutant capsids from sucrose fraction No. 7; diameter
30 nm in average.
22
3.3 Assembly tendency difference between WT and D2ND4N dimers
For core protein assembly, its process is always affected by protein concentration,
ionic strength and pH. Since the experiments are all conducted in buffer N at pH=9.6, we
just focus on protein and salt concentration effect.
Proteins of different concentrations were used to test the critical concentration for
capsid formation. By using DLS, the success of assembly was determined by the obvious
increment in scattered light intensity. Their extent of assembly of can also be estimated
from the rate of intensity increment.
In the assembly study, we observed that mutant dimers are more difficult to form
capsids compared to WT dimers. When titrating KCl into WT and mutant dimer solution
of similar concentrations, the self-assembly from mutant dimers requires more salt to be
triggered (figure 16a). At WT final concentration of 1.301 mg/ml (after capsid assembly)
and D2ND4N final concentration of 1.4781 mg/ml, WT needs more than 0.8 M K+ while
D2ND4N requires more than 1 M K+ to cause the scattered light increment, indicating the
start of protein aggregation. Also, WT protein assembly can be triggered at lower protein
concentration: 1.266 mg/ml WT (final concentration) is enough for detectable WT capsid
formation, while D2ND4N needs to reach around 1.358 mg/ml (final concentration). The
intensity increment is faster for WT than D2ND4N, which means more WT capsids can
be formed with similar amount of salt addition (figure 16b). The easier attraction for WT
Cp149 may be due to more charges it carries than D2ND4N, making stronger counterion
mediated attraction during the capsid formation process.
23
a
b
Figure 16: WT and D2ND4N capsid formation tendency comparison. (a). D2ND4N
dimer solution needed more than 1M K+ to form capsid, while WT dimer just needed less
than 0.83 M K+ to show intensity increment. (For sample in this plot: WT protein final
concentration is 1.301 mg/ml and D2ND4N is 1.4781 mg/ml). (b). When size
contribution to intensity is excluded, WT solution always brought more obvious intensity
24
growth with addition of similar amount of salt, compared to D2ND4N solution, which
means more WT capsids were formed. (Scattered intensity I ∝ c*M, and capsid molecular
weight M ∝ Rh2, so I ∝ C*Rh2. Rh2 can be divided from intensity to exclude the capsid size
contribution difference on intensity).
25
CHAPTER IV
CONCLUSION
In summary, wild-type capsid protein Cp149 and its mutant D2ND4N with less
charge were both triggered to assemble into capsids with K+ in buffer N at room
temperature. It was found that mutant dimers are less favored to form into capsids, with a
higher critical protein concentration and higher demand of salt, may be due to less
attraction force compared to WT Cp149. Besides, two assembled capsids exhibit different
average sizes from DLS CONTIN results and different sediment coefficients, which may
indicate slight difference in capsid structure or T=3/T=4 component and need to be
further proved by other experiments like 3D reconstruction analysis.
26
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2
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