Cargo Retention inside P22 Virus-Like Particles

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES

Article

Cargo Retention inside P22 Virus-Like Particles Kimberly McCoy, Ekaterina Selivanovitch, Daniel Luque, Byeongdu Lee, Ethan Edwards, Jose R. Castón, and Trevor Douglas Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00867 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 9, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Cargo Retention inside P22 Virus-Like Particles

1 2

Kimberly McCoya, Ekaterina Selivanovitcha, Daniel Luquec,d, Byeongdu Leeb, Ethan

3

Edwardsa, Josè R. Castónc, Trevor Douglasa*

4 5

a

6

IN 47405, USA

7

b

8

South Cass Ave., Argonne, IL 60439, USA

9

c

Department of Chemistry, Indiana University, 800 East Kirkwood Ave., Bloomington,

X-ray science division, Advanced Photon Source, Argonne National Laboratory, 9700

Department of Structure of Macromolecules, Centro Nacional de Biotecnología

10

(CNB–CSIC), Darwin 3, 28049 Madrid, Spain.

11

d

Centro Nacional de Microbiología/ISCIII, 28220 Majadahonda, Madrid, Spain

12 13

To whom correspondence should be addressed.

14

E-mail: [email protected], tel. (812) 856-6936

15 16

Abstract

17

Viral protein cages, with their regular and programmable architectures, are excellent

18

platforms for the development of functional nanomaterials. The ability to transform a

19

virus into a material with intended structure and function relies on the existence of a

20

well-understood model system, a non-infectious Virus-Like Particle (VLP) counterpart.

21

Here, we study the factors important to the ability of P22 VLP to retain or release

22

various protein cargo molecules depending on the nature of the cargo, the capsid

23

morphology, and the environmental conditions. Because the interaction between the

ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

internalized Scaffold Protein (SP) and capsid Coat Protein (CP) is non-covalent we

2

have studied the efficiency with which a range of SP variants can dissociate from the

3

interior of different P22 VLP morphologies and exit by traversing the porous capsid.

4

Understanding the types of cargos that are either retained or released from the P22

5

VLP will aid in the rational design of functional nanomaterials.

6 7

Keywords (4-6): virus-like particle, cargo release, P22 VLP, bioinspired, nanomaterial,

8

protein cage

9

Introduction

10

Viruses, usually considered undesirable disease-causing pathogens, are a source of

11

biomaterials and are increasingly the inspiration for the creation of functional

12

nanomaterials. Virus-like particles (VLPs) are the non-infectious counterparts of viruses

13

and are often highly regular, nanometer-sized structures that self-assemble from

14

multiple copies of a small number of molecular components via many weak, non-

15

covalent and thus reversible interactions. The nature of this assembly results in dynamic

16

aspects of the VLP that have been successfully leveraged for the directed self-assembly

17

and encapsulation of a wide range of non-native cargos within VLPs.1-3 Studies on VLPs

18

have highlighted the ability to encapsulate cargo but are generally static assemblies, not

19

often designed to release their cargo. Viruses, on the other hand, naturally encapsulate

20

and protect a sequestered cargo and release this cargo, which is usually associated

21

with capsid disassembly or cargo ejection through the relief of large internal pressures.4,

22

5

23

critical for the design of new, functional nanomaterials. Here, we investigate the

Understanding the mechanisms of cargo encapsulation and release in VLP systems is


Page 2 of 36

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

interactions between the coat protein (CP) and internalized scaffolding proteins (SP) in

2

different morphologies of the P22 VLP by monitoring SP retention and release from the

3

capsid when the charge and size of the SP are altered (i.e. through SP truncation or

4

through fusion of cargo to the SP).

5 6

The P22 VLP is a well-developed protein-cage model system that has been studied

7

both as a nanomaterial in the context of cargo encapsulation,6-9 molecular

8

presentation,10-12 and higher order assembly13-15 as well as a model for understanding

9

the physical aspects of structure and function of the infectious bacteriophage.16-18

10

Assembly of the VLP occurs through co-polymerization of 420 coat proteins (CP) and

11

100-300 internalized scaffold proteins (SP) and results in a monodispersed population

12

of T = 7 procapsids (PC). SPs exit the capsid likely through hexameric pores during

13

DNA packaging in the bacteriophage, or during heat treatment (to 65 °C) of VLPs, both

14

of which result in an irreversible morphological expansion of the PC. During formation of

15

this expanded (EX) capsid the conformation of the CP subunits are altered and the

16

putative SP-binding site on the interior of the capsid is no longer accessible.19 Further

17

heating VLPs to 75 °C leads to the release of the 12 pentamers of the icosahedron,

18

yielding a more rounded wiffleball (WB) capsid with approximately 10 nm pores, but with

19

a similar size and CP conformation as seen in the expanded capsid.20 The retention of

20

SP variants inside of P22 therefore depends on the morphology of P22, which

21

determines whether the putative SP binding site is accessible and also whether the pore

22

size of the capsid is large enough to allow cargo to traverse the capsid wall.



1

The essential residues in the 303-amino acid wild type SP that are responsible for CP

2

binding are located in a C-terminal helix-turn-helix motif.21 In particular, R293 and K296

3

on the SP22 and residues (D14 and E18) on the CP23 are essential for the co-assembly

4

into the P22 PC VLPs. While the rest of the SP structure is unsolved, it is not

5

necessarily intrinsically disordered. In solution it adopts a U-shaped helix-turn-helix, with

6

its N- and C-terminus in close proximity and has a diameter of 2.2 nm and a length ~2.5

7

nm.24, 25 The SPs remain bound to the interior surface of the PC particle but can be

8

removed with low concentrations of chaotrope (0.5 M urea or guanidine•HCl) to

9

generate the empty shell (ES) morphology.26, 27 Incubation of the ES, once the

10

chaotrope is removed, with an excess of SP results in the formation of ‘stuffed shells’

11

indicating that the SP can re-enter and re-bind to the empty capsid.28, 29 There are two

12

distinct steps involved in the process whereby SP escapes from PC; SP dissociation

13

from CP followed by escape through the capsid pores. We have measured these two

14

steps in combination by observing the decrease in molecular weight of the VLP after

15

cargo leaves the capsid. Because SP-CP binding is largely electrostatic, we also test

16

whether this cargo loss behavior is altered under different ionic strength conditions.

17 18

A truncated version of the SP (residues 142-303; SPt), which retains the essential

19

residues for binding CP,30 has been shown to direct the assembly of T = 7 P22 VLP

20

capsids. The full-length, wild type SP (SPwt) has a pI of 5.2 while SPt has a pI of 9.2,

21

resulting in a highly cationic protein at neutral pH. Because the interior of the capsid is

22

highly negatively charged,31 we compared differences in protein retention in P22 VLPs

23

between SPwt and SPt. We also probed the retention of a previously studied enzyme-


Page 4 of 36

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

SPt fusion in the P22 wiffleball capsid, which exhibits similar size dimensions to the

2

wiffleball pores.32 While the enzymatic activity of this tetrameric glucosidase (CelB) has

3

been studied inside of P22 wiffleball, it is not known whether the large capsid pores

4

coupled with capsid dynamics allow cargo to leak out over time.

5 6

Here we investigate the cargo loss behavior of various encapsulated molecules from the

7

P22 VLP as a function of time, P22 morphology, temperature, and ionic strength using

8

multi-angle light scattering, small angle x-ray scattering, and cryo-electron microscopy.

9

We show that the release or retention of cargo packaged inside the P22 VLP through

10

non-covalent interactions is dependent on both electrostatic interactions and the

11

relationship between cargo size and the pore size of the capsid. This suggests new

12

approaches for the engineering of synthetic VLP materials with controlled release

13

capabilities.

14 15

Experimental

16

Particle Preparation

17

P22wt and P22 CelB

18

E. coli strains either with 1) incorporated pET expression vector containing the P22 Coat

19

Protein (CP) and the P22 Scaffolding Protein33 or 2) incorporated pET duet expression

20

vector containing the P22 Coat Protein (CP) and the truncated SP fused to the C-

21

terminus of CelB (NdeI/XhoI)32 were grown in LB medium at 37 °C in the presence of

22

ampicillin (50 µg/mL) to maintain selection for the plasmid. Protein expression was

23

induced by addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a final



1

concentration of 0.3 mM once the cells reached mid-log phase (OD600=0.6). Cultures

2

were grown an additional 4 hours after induction with IPTG, and then the cells were

3

harvested by centrifugation at 4,500 x g, re-suspended in minimal PBS, and stored at -

4

80 °C.

5

The E. coli cell solution was thawed at room temperature then diluted in lysis buffer (50

6

mM sodium phosphate 100 mM sodium chloride pH 7) at a ratio of 5-10 mL lysis buffer

7

per g of cell pellet. After incubating with DNase, RNase, and lysozyme with gentle

8

rocking for 30 minutes at room temperature, cells were lysed via sonication. The

9

solution was centrifuged at 12,000 x g for 45 minutes to remove cellular debris then

10

centrifuges at 45,000 rpm through a 35% sucrose cushion. Pelleted particles were re-

11

suspended in minimal lysis buffer and then applied to a Sephacryl-500 (GE Healthcare

12

Life Sciences) size exclusion column using a Biorad Biologic Duoflow FPLC. Fractions

13

containing P22 VLPs were pooled and concentrated via ultracentrifugation.

14 15

6xH-SPt and GFP-SPt-6xH

16

E. coli strains with either 1) incorporated pRSF duet expression vector containing the

17

P22 Scaffolding Protein truncated version (residues 142-303) or 2) incorporated pRSF

18

duet expression vector containing the truncated SP fused to the C-terminus of GFP

19

were grown in LB medium at 37 °C in the presence of kanamycin (30 µg/mL) to

20

maintain selection for the plasmid. Protein expression was induced by addition of

21

isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM once the

22

cells reached mid-log phase (OD600=0.6). Cultures were grown an additional 4 hours


Page 6 of 36

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

after induction with IPTG, and then the cells were harvested by centrifugation at 4,500 x

2

g, re-suspended in minimal PBS, and stored at -80 °C.

3 4

The E. coli cell solution was thawed at room temperature then diluted in lysis buffer (50

5

mM sodium phosphate 100 mM sodium chloride pH 7.0 at a ratio of 5-10 mL lysis buffer

6

per g of cell pellet. Samples were incubated with DNase, RNase, lysozyme, a Pierce

7

Protease Inhibitor tablet for 30 minutes at room temperature with gentle rocking, and

8

then cells were lysed via sonication. Solution was centrifuged at 12,000 x g for 45

9

minutes to remove cellular debris, then filtered through a 0.45 micrometer filter.

10

Samples were loaded onto a 5 ml Roche Ni-NTA column at a flow rate of 1 ml/min. The

11

column was washed with 5 mM imidazole to remove non-specific binders then protein

12

was eluted using a gradient of imidazole (10 mM – 500 mM). The presence of pure

13

protein in fractions was determined by SDS-PAGE analysis and appropriate fractions

14

were combined. Immediately before use in in vitro assembly, samples were dialyzed out

15

of imidazole buffer.

16 17

ES prep for CP

18

P22 PCs were incubated on a rocker in 0.5 M GuHCl for 1 hour at 4 °C. Capsids were

19

pelleted in ultracentrifuge at 45,000 rpm (F50L-8x39 rotor) for 50 min followed by

20

resuspension in PBS and then incubation in 0.5 M GuHCl for 1 h. This extraction

21

process was repeated a total of three times to remove SP, which was verified by SDS-

22

PAGE. ES were dissociated into free CP by mixing ES:6M GuHCl in a 1:1 ratio for a

23

final concentration of 2 mg/ml CP in 3 M GuHCl.



1 2

In vitro assembly

3

P22-SPt and P22-GFP were separately assembled by mixing CP subunits (∼2 mg/ml,

4

as prepared above in 3 M GuHCl) with wither purified SPt or GFP-SPt at a 1:1 molar

5

ratio. The volume of each cargo was adjusted such that the final concentration of GuHCl

6

was 1.5 M in all assembly reaction mixtures. The mixture was dialyzed into assembly

7

buffer (50 mM Tris-HCl, 25 mM NaCl, 2 mM EDTA, 3 mM β-mercaptoethanol, and 1%

8

glycerol) for 12–18 h. Samples were centrifuged at 21,000 x g for 5 minutes to removed

9

aggregated protein then centrifuged at 45,000 rpm to concentrate capsids and to

10

remove soluble protein not associated with the capsid.

11 12

Expansion

13

P22 and P22-CelB were each heated to 67 °C for 20 minutes, then cooled at RT.

14

Samples were centrifuged at 21,000 x g for 5 minutes to removed aggregated protein

15

then centrifuged at 45,000 rpm to concentrate capsids and to remove soluble protein

16

not associated with the capsid.

17 18

0.5 mg/ml P22-GFP and P22t were each reacted with 0.1% sodium dodecyl sulfate

19

(SDS) for 15 minutes and then centrifuged at 45,000 rpm to remove SDS.

20 21

Wiffleball

22

All P22 constructs capsids were expanded to the wiffleball morphology by heating in a

23

75 °C water bath for 20 minutes, then cooled at RT. Samples were centrifuged at


Page 8 of 36

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

21,000 x g for 5 minutes to removed aggregated protein then centrifuged at 45,000 rpm

2

to concentrate capsids and to remove soluble protein not associated with the capsid.

3 4

Particle Characterization

5

SDS-PAGE

6

Protein samples were mixed with 4X SDS loading buffer containing DTT and denatured

7

in a 100°C water bath for 5 minutes. Samples were loaded onto a 12% acrylamide

8

resolving gel (5% acrylamide stacking gel) and separated using a constant current of 35

9

milliamperes for approximately 1 hour. Gels were stained with either or InstantBlue

10

(Expedion) or with Coomassie blue stain then destained. Images were taken on a UVP

11

MultDoc-IT Digital Imaging System.

12 13

Agarose gel

14

10 µL each sample at ~1 mg/mL was mixed with 5 µL non-denaturing protein loading

15

dye and loaded into a 0.8% agarose gel. Constant voltage of 65 V was applied for 3

16

hours. Gels were stained and imaged as described above.

17 18

SEC-MALS

19

Samples were separated using a WTC-200S5 (Wyatt Technologies) size exclusion

20

column utilizing an Agilent 1200 HPLC at a 0.7 mL/min flow rate in 50 mM phosphate

21

100 mM Sodium Chloride and 200 ppm Sodium Azide pH 7.2 buffer. For each sample,

22

three 25-µL injections were loaded onto the column with a total run time of 30 minutes.

23

Samples were detected using a Wyatt HELEOS Multi Angle Laser Light Scattering



1

(MALS) detector and an Optilab rEX differential refractometer (Wyatt Technology

2

Corporation). The average molecular weight, MW, was calculated with Astra 5.3.14

3

software (Wyatt Technology Corporation) based on the molecular weight distribution

4

and using the refractive index increment (dn/dc) 0.185.

5 6

TEM

7

5 µL of each sample (diluted in water to 0.1-0.3 mg/mL) was applied to a glow

8

discharged formvar coated grid (Electron Microscopy Sciences). After 30 seconds

9

excess liquid was wicked away with filter paper then immediately stained with 5 µL 2%

10

uranyl acetate. Excess stain was wicked away with filter paper after 25 sec then allowed

11

to air dry. Images were taken on a JEOL 1010 transmission electron microscope at an

12

accelerating voltage of 100 kV.

13 14

SAXS

15

Small angle x-ray scattering data were collected at the Advanced Photon Source at

16

Argonne National Laboratory at beam line 12-ID-B with a 14 keV x-ray beam. Samples

17

were continuously moved using a syringe pump system to minimize beam damage and

18

subjected to a 1 sec. exposure time, 2 sec. interval, and 20 total shots per sample and

19

detected by a Pilatus 2M detector. The scattering angle was calibrated using silver

20

behenate as a standard. 2D images were then converted to 1D curves, and the 20

21

curves for each sample were then averaged. The background due to the sample buffer

22

was measured separately and subtracted from the averaged data. The pair distance


Page 10 of 36

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

distribution P(r) function was obtained by fitting the experimental form factor P(q)

2

scattering of P22 PC in solution using the software GNOM (Figure 2b).

3 4

Cryo-EM

5

Particle measured were either P22-CelB (described above) or P22 S39C.34 Samples (5

6

µl) were applied to Quantifoil R 2/2 holey grids, blotted, and plunged into liquid ethane.

7

Cryo-EM images were recorded, under low-dose conditions, with a FEI Eagle CCD

8

camera in a Tecnai G2 electron microscope equipped with a field emission gun

9

operating at 200 kV and at a detector magnification of 69,444X (2.16 Å/pixel sampling

10

rate).

11 12

General image processing operations were performed using Xmipp,35 and graphics

13

representation were produced by UCSF Chimera.36 Xmipp automatic picking routine

14

was used to select 2,787, 9,639 and 12,660 P22 Empty PC, WB and CelB-WB particles,

15

respectively. Defocus was determined with CTFfind37 and CTF phase oscillations were

16

corrected in the images by flipping them in the required lobes. Homogeneous

17

populations were selected by 2D classification using the Xmipp CL2D reference-free

18

clustering routine.38 The published structures of P22 PC and EX particles (PDBs

19

2XYY31 and 5UU5,39 respectively) were filtered to 30 Å, size-scaled and used as initial

20

models for PC and WB samples, respectively. Xmipp iterative Projection Matching

21

routine40 was carried out to determine and refine the origin and orientation of each

22

particle and after iterative refinement 2,010, 4,488 and 11,041 P22 Empty-PC, WB and

23

CelB-WB particles, respectively, were included in the final 3DRs and resolution was



1

assessed by FSC between independent half-dataset maps. Applying a correlation limit

2

of 0.5 (0.3), the resolution for the P22 Empty-PC, WB and CelB-WB maps was 19.6

3

(17.0), 18.5 (16.4) and 13.9 (12.3), respectively. The 3D reconstructions have been

4

deposited in the Electron Microscopy Data Bank (www.ebi.ac.uk/pdbe/emdb) with

5

accession nos. EMD-4387 (PC Empty), EMD-4388 (WB) and EMD-4389 (CelB-WB).

6 7

Results and Discussion

8

We investigated a range of SP variants to determine which could exit the capsid after

9

dissociating from the putative binding site on the CP. The average molecular weight

10

(MW) of P22 particles was determined using size exclusion chromatography coupled to

11

multi-angle light scattering (SEC-MALS). The intensity of scattered light is directly

12

proportional to the average particle MW, which can be calculated when the concentration

13

of protein and the refractive index increment (dn/dc) are known. The MW of purified P22

14

VLPs (PC morphology) was determined for particles eluting at approximately 14

15

minutes from the SEC column, which is characteristic of P22 PC particles. After

16

subtracting the expected mass contribution of 420 CPs from the measured MW, the

17

average number of cargo molecules per PC could be calculated (using SP molecular

18

weight of 33.4 kDa). Immediately following purification, P22 PCs that were assembled

19

with wild type SPwt were analyzed by SEC-MALS (Figure 1a) and found to contain an

20

average of 130 ± 3.9 SPwt per particle (Table 1). To exit the PC, SP must first dissociate

21

from the putative binding site within the PC and diffuse out of the capsid, likely through

22

the ~ 2.5 nm pores at the hexamer centers.28

23


Page 12 of 36

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Disruption of the putative SP-binding site occurs during the irreversible conversion of

2

PC to expanded (EX) capsids due to a rearrangement of the subunit conformation.

3

Purified PC VLPs were heated to 65 ºC for 10 minutes and the transformation to the EX

4

morphology was verified by a mobility shift on a native agarose gel and by an increase

5

in the particle radius measured by MALS (Figure 1b; Table 1). The PC to EX conversion

6

reduced the P22 MW from 23.9 ± 0.2 MDa (for the PC) to 20.5 ± 0.1 MDa (for the EX),

7

which is close to the theoretical value of 19.6 MDa for a capsid consisting solely of 420

8

CPs. The additional 0.9 MDa can be attributed to some residual SPwt still bound to the

9

EX capsid, which was verified by SDS-PAGE (Supp. Figure S1). Further heating of the

10

EX to obtain the wiffleball (WB) morphology resulted in capsids with an average MW of

11

18.3 ± 0.1 MDa, which is higher than the theoretical 16.8 MDa for P22 WB capsids

12

containing 360 CPs, in which 12 pentamers have been removed. SDS-PAGE analysis

13

of this sample showed no remaining SPwt thus the higher MW might be due to

14

incomplete ejection of all of the pentamers from the capsid (Supp. Figure S1). This

15

result is consistent with previous findings, which showed capsids heated to 75 °C to

16

obtain the WB morphology exhibited an average MW of 18.3 MDa.41 Further evidence

17

for the retention of pentamers in WB samples was observed using cryo-electron

18

microscopy (cryo-EM) and image reconstruction. A 3D reconstruction (3DR) was

19

calculated for P22 WB capsids (Figure 1d). Based on a Fourier shell correlation (FSC)

20

coefficient, the resolution achieved was 19.4 Å (for the 0.5 threshold) or 15.8 Å (for the

21

0.3 threshold) (Supp. Figure S2). After icosahedral averaging was performed, density

22

was observed at the 12 5-fold symmetry sites, albeit at a lower intensity than the rest of

23

the capsid, suggesting that not all pentamers are removed in WB samples derived from



1

VLPs assembled with full-length SPwt (Figure 1d, bottom right) and consistent with the

2

slightly higher than expected mass observed by MALS.

3

4 5

Figure 1. P22 assembled with full length SPwt. a) SEC-MALS of PC, EX, and WB with

6

molecular weight traces. b) Native agarose gel of PC, EX, and WB. c) CryoEM of P22

7

WB, Scale bar, 50 nm. d) Surface-shaded representation of the outer (top, left) and

8

inner (top, right) surfaces of P22 WB from 3DR, viewed along an icosahedral twofold

9

axis. The map is contoured at 1σ above the mean density. An 8.0-nm-thick P22 WB slab

10

countered at 1σ above the mean density. Icosahedral symmetry axes are numbered

11

(bottom, left). Central sections from the 3DRs of EX (left) and WB (right) P22 capsids.

12

Compare the density difference at the five-fold axes (arrows).

13 14

The CP-SP interaction can also be disrupted by treating PC capsids with 0.5 M

15

guanidine hydrochloride or 0.5 M urea, and these treatments result in the removal of


Page 14 of 36

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

most or a subset of SPwt, respectively (Supp. Figure S3). This difference in the amount

2

of SPwt released suggests that the effects of both the chaotrope and ionic strength

3

contribute to the removal of the SPwt. We observed that even in the absence of heat or

4

chemical treatment, SPwt are slowly released from the P22 PC capsid over time. To

5

monitor this, and to investigate the role of ionic strength in the removal of SPwt, we

6

measured the molecular weight of capsids kept at room temperature in buffer (50 mM

7

phosphate, 200 ppm NaN3) in various salt concentrations (20, 200, or 1000 mM NaCl)

8

by MALS over a period of 58 days starting immediately following purification of the PC.

9

In buffer containing 20 mM NaCl, the number of SPwt decreased from 130 ± 3.9 to 58.2

10

± 1.8 SPwt over a 58-day period (Figure 2a). However, the hydrodynamic radius and

11

capsid morphology remained the same, suggesting the capsid structure remained

12

unchanged over this time period (Table 1; Supp. Figure S4). In the first 4 days (96 hrs)

13

of monitoring, SPwt exited P22 at a rate of 0.30 SPwt/hr after which the release rate

14

slowed. Data corresponding to SPwt release fit well to an exponential decay fit when the

15

last data point was excluded (1392 hours), suggesting biphasic release behavior. This

16

result is consistent with the presence of both high and low affinity sites which have been

17

described for SP-CP binding.28, 29 Our data suggests that a lower affinity population of

18

SPwt are released first from P22 and the more tightly bound population is released at a

19

slower rate. Additionally, because protein samples are usually stored at 4 °C, we

20

compared the rate of SPwt release at room temperature to release at 4 °C. As expected

21

the release rate at 4º C was slower at 0.12 SPwt/hr in the first 4 days (96 hrs) of

22

monitoring (Supp. Figure S5).

23



1

2 3

Figure 2. SPwt retention in P22 as a function of time and ionic strength. a) The rate at

4

which SPwt escape the capsid is correlated to the ionic strength of the buffer. b) Pair-

5

distance distribution function from SAXS of individual particles in solution comparing

6

purified PC at t = 0 (blue) and t = 30 (red) days, and empty shells (black). c) Cryo-EM

7

image of empty P22 PC. Scale bar, 50 nm. d) Outer surface of the empty capsid,

8

viewed along a twofold axis of icosahedral symmetry. The map is contoured at 1σ above

9

the mean density. e) Central sections from the 3DR of P22 PC with (left) and without

10

(right) SPwt. The two protein shells are virtually identical except for the presence of SPwt-

11

related densities (left, arrows). f) Central section of empty PC with docked CP. In

12

comparison with fresh P22 PC, no unoccupied density is left after CP docking,

13

indicating the absence of SPwt.

14


Page 16 of 36

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

When P22 capsids were transferred to buffer containing 200 mM sodium chloride, after

2

purification, the initial average number of SPwt packaged inside the PC was 116 ± 18,

3

which is less than that of capsids in the lower ionic strength buffer. This 200 mM NaCl

4

sample originated from the same initial stock as capsids in 20 mM NaCl buffer,

5

suggesting that the increased ionic strength caused the immediate loss of some SPwt

6

from the P22 capsid. SPwt were subsequently released at a similar rate (0.25 SPwt/hr),

7

over the first 4 days after the initial measurement (96 hrs), as P22 samples in buffer

8

containing 20 mM NaCl. After the 58-day monitoring period, the average number of SPwt

9

under these elevated ionic strength conditions decreased to 41.2 ± 0.3 SPwt per particle

10

while the particle size (Rh) and morphology remained the same (Table 1; Supp. Figure

11

S4). P22 capsids that were suspended in buffer at even higher ionic strength, 1 M NaCl,

12

after purification showed a similar trend in which the initial amount of encapsulated SPwt

13

was lower (101 ± 3.0 SPwt per P22) than capsids in either 20 mM or 200 mM NaCl.

14

Additionally, the rate at which SPwt exit P22 under these conditions in the first 4 days

15

(96 hrs) of monitoring was approximately double that of SPwt in capsids in either 20 mM

16

or 200 mM NaCl (0.5 SPwt/hour). The final number of SPwt (12.2 ± 0.3) after 58 days

17

was less than both samples at lower ionic strength with no change in capsid size or

18

morphology (Table 1; Supp. Figure S4). These data, showing that increase in ionic

19

strength results in more SPwt being released from P22, is likely due to screening of

20

electrostatic interactions known to be important for CP-SP binding. Thus, wild type SP

21

slowly leaks out of P22 PC after dissociation from the putative binding site and

22

increasing the ionic strength increases the rate at which SPwt are released from the

23

capsid.



Page 18 of 36

1 2 3 4 5 6 7

Table 1. Molecular weight and radius of hydration values for P22.

Construct:

Radius of

Molecular

[NaCl]: Day

hydration (Rh)

Weight (MDa)

measured

(nm)

PC: 20mM: D1

25.9 ± 0.1

23.9 ± 0.2

PC: 20mM: D58

25.9 ± 1.1

21.5 ± 0.1

PC: 200mM: D1

26.2 ± 0.2

23.5 ± 0.6

PC: 200mM: D58

25.2 ± 0.2

21.0 ± 0.01

PC: 1000mM: D1

25.7 ± 0.2

23.0 ± 0.1

PC: 1000mM: D58

24.7 ± 0.1

20.0 ± 0.01

EX

28.2 ± 0.2

20.5 ± 0.1

WB

28.2 ± 0.1

18.3 ± 0.1

8 9

To investigate the structures of the P22 PC over this experiment, the samples were

10

interrogated by small angle X-ray scattering (SAXS). Pair-distance (P(r)) distribution

11

functions, which evaluate the distribution of electron density within a capsid, were

12

acquired from SAXS measurements. These data confirm that a change occurred in the


Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

electron distribution in the capsid after 30 days (PC30). Figure 2b shows normalized P(r)

2

distributions for freshly purified capsids (PC), ES, and PC30. ES shows a characteristic

3

P(r) function of an object with an empty core, which exhibits an asymmetric peak with a

4

maximum at approximately 48.2 nm. This peak maximum is followed by a steep drop-off

5

and reaches zero at approximately 60 nm, which is in agreement with the diameter of

6

the ES and PC morphologies of P22. PC capsids, however, have a maximum intensity

7

of 34.6 nm and show a more Gaussian profile that is characteristic for solid spheres,

8

indicating the presence of mass on the interior. PC30 capsids kept at room temperature

9

show a profile that is a linear superposition of P(r) functions of PC and ES, suggesting

10

that some SPwt remains encapsulated, which is consistent with MALS data, with an

11

estimated 10% of SPwt retained inside of P22 (Supp. Figure S6). Importantly, the overall

12

particle diameter does not change, indicating that the observed changes to the particle

13

morphology measured by SAXS are due to the loss of mass from the capsid interior. 3D

14

cryo-EM analysis of P22 PC kept at 4 ºC for approximately 6 months confirmed our

15

biochemical analysis; these capsids were empty and lacked SPwt-related densities

16

(Figure 2c-e). The particle radius of empty PC was 29.8 nm (estimated from the radial

17

density profile from the 3DR), in concordance with the outer radius of fresh PC that

18

contained SPwt.42 Docking analysis of the P22 CP model [Protein Data Bank (PDB) ID

19

2XYY] in the empty PC confirmed that, after CP docking, no extra density was

20

unoccupied, indicating SPwt were unbound (Figure 2f).

21 22

To investigate the behavior of larger cargos encapsulated within the P22 we measured

23

the MW of P22 PC encapsulating GFP or the enzyme CelB on the interior of P22.32, 41



1

These protein cargos are genetically fused to a truncated form of the SP where the first

2

141 residues have been removed from the N-terminus. GFP-SPt (46.5 kDa),

3

encapsulated inside of P22 using an in vitro method (see methods), is a monomer and

4

has been shown to exit the WB capsid through the 10 nm capsid pores, which we

5

verified here. CelB-SPt (73.5 kDa) is a tetrameric β-glucosidase and we have previously

6

measured the kinetic properties in the PC, EX, and WB32 morphologies of P22-CelB as

7

well as the structural properties in the PC and EX morphologies.42 Because CelB has

8

similar size dimensions to the WB pores, we investigated whether it could exit the

9

capsid over time. In the PC morphologies both P22-GFP and P22-CelB maintain the

10

same MW over a period of 35 and 22 days respectively suggesting that the folded

11

structures of GFP-SPt and CelB-SPt are effectively larger than the ~2.5 nm PC pores

12

(Figure 3a) and cannot leave the capsid. Treatment with 0.5 M GuHCl yielded capsids

13

with similar molecular weights indicating that size constraints alone, and not SPt-CP

14

binding retains these two cargo proteins (Supp. Figure S7).

15


Page 20 of 36

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Figure 3. Cargo-SPt retention in P22 PC and WB. a) The number of CelB-SPt or GFP-

2

SPt encapsulated inside of P22. b) SDS-PAGE gel of soluble and aggregated portions

3

of P22-CelB after expansion. c) Cryo-EM image of P22-CelB WB. Scale bar, 50 nm. d)

4

Surface-shaded representation of the outer (left) and inner (center) surfaces, viewed

5

along an icosahedral twofold axis, of P22-CelB WB. The map is contoured at 1σ above

6

the mean density. CP shell is yellow; cargo density is red. An 8.0 nm-thick P22-CelB

7

WB slab countered at 1σ above the mean density (right). e) Central sections from the

8

3DRs of EX (left) and CelB WB (right) P22 capsids. Compare the density difference at

9

the five-fold axes (arrows). f) Radial density profiles of 3DR of empty (WB, dashed line)

10

and CelB-loaded WB (CelB WB, solid line). WB shells are essentially superimposable.

11 12

When the capsids were expanded to form the EX morphology (Supp. Figure S8) some

13

material aggregated and precipitated from solution in both the GFP and CelB samples.

14

Immediately after expansion the soluble P22-GFP EX was measured to have a MW of

15

24.6 ± 0.1 MDa (down from 30.5 ± 0.1 MDa prior to expansion) and the P22-CelB EX

16

MW was 26.4 ± 0.3 MDa (down from 30.7 ± 0.3 MDa prior to expansion) (Supp. Table

17

S1). SDS-PAGE data showed that the aggregated protein formed during the expansion

18

was composed of roughly the same mixture of CP and CelB-SPt proteins (Figure 3b) as

19

the soluble protein. This suggests that whole capsids, and not just escaped cargo, were

20

aggregating and causing the observed decrease in P22-CelB MW. It is possible that

21

some degree of pentamer loss could contribute to MW decreases but because the

22

difference is 4.3 MDa and pentamers only contribute 2.8 MDa to the capsid MW, this

23

cannot be the only factor. The apparent decrease in MW observed during expansion of



1

P22 has been previously observed41, 42 and is likely due to the removal of a

2

subpopulation of higher molecular weight, perhaps misassembled capsids, and not due

3

to cargo escaping from P22. However, hydrodynamic radii for each PC is consistent

4

with wt PCs, suggesting capsids that are removed from the population during expansion

5

do not have a significantly larger diameter than normal PCs. P22-CelB PC capsids

6

heated to 60 °C (7 °C below the temperature required for expansion of this construct)

7

showed negligible protein aggregation and had the same MW before and after heating

8

(30.9 ± 0.1 and 30.5 ± 0.1 MDa, respectively; Supp. Table S1), suggesting particles are

9

stable up to at least 60 °C. It is possible that changes in the mechanical properties due

10

to cargo packaging alter the ability of the capsid to expand, therefore capsids with more

11

cargo may be less likely to form bona fide and stable EX particles. Indeed, packaging of

12

non-native cargo increases the osmotic pressure inside the capsid and the particle

13

rigidity, which could result in some disassembly during expansion.42 Another possible

14

explanation is that there are imperfect capsids present, possibly formed due to

15

encapsulation of larger cargos, which co-purify with T = 7 capsids but aggregate upon

16

heat treatment required for expansion.

17 18

When heated to 75 °C both P22-GFP and P22-CelB samples showed a shift in mobility

19

on an agarose gel (Supp. Figure S8), consistent with the formation of WB capsids. As

20

shown in Supplemental Table 1, P22-GFP samples decreased in MW to 15.7 ± 0.02

21

MDa, close to the expected MW of empty WB capsids (16.8 MDa) after this heat

22

treatment. However, the ratio of absorbance due to GFP and total protein absorbance

23

(492nm/280nm) decreased from 0.48 to 0.16, suggesting the retention of a population


Page 22 of 36

Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

of GFP (Supp. Table S2). It is possible therefore that the average molecular weight in

2

the P22-GFP WB sample is a combination of a residual population of GFP-SPt proteins

3

and less than the expected 360 CP in WB. It is also possible that positively charged

4

GFP-SPt are interacting with the exterior of the negatively charged P22 capsid. Future

5

studies may investigate the capsid structure and stability of these P22 VLPs. The

6

difference in mass between P22-CelB EX to WB capsids also showed a decrease from

7

26.4 ±0.2 to 25.5 ± 0.05 MDa. The expected mass difference between EX and WB is

8

2.8 MDa, however, only a 0.9 MDa decrease was observed, suggesting that not all of

9

the pentamers were removed in P22-CelB WB. Alternatively, a subset of pentamers

10

may have been removed during the conversion of P22-CelB PC to EX, thus the MW

11

difference from EX to WB would be less than the expected 2.8 MDa. 3D cryo-EM

12

analysis indicated that P22-CelB WB mostly lacked pentamers at the fivefold axes

13

(Figure 3c, d).

14 15

Compared to empty WB capsids, P22-CelB WB capsids contained internal density

16

corresponding to CelB-SPt. These results suggest that the cargo may play a role in the

17

ejection of pentamers, as empty WB capsids retained density at pentameric sites

18

(Figure 1d). Following conversion to WB, no subsequent change in MW was observed

19

for the CelB WB over a 22-day period, suggesting that the CelB cargo does not leave

20

the capsid over time (Figure 3a, triangles). Comparison of the average radial densities

21

of empty WB and CelB WB capsids (obtained after imposing icosahedral symmetry)

22

indicated that the cargo is present along the internal volume of the latter (Figure 3f).

23

Because the expected dimensions of a CelB tetramer (10.1 nm × 10.1 nm × 5.7 nm 42)



1

are similar to the presumed ~10 nm pore size of P22 WB, it is likely that the cargo is

2

sterically hindered from traversing the capsid pores and thus these cargo proteins are

3

retained.

4 5

The cargo encapsulation of GFP and CelB is predicated on their genetic fusion to a

6

truncated form of the scaffolding protein (SPt) in which the first 141 residues have been

7

removed. This minimal form of the scaffold protein is still able to direct the assembly of

8

capsids and has been the minimal SP motif to which a wide range of cargos have been

9

attached for the directed assembly of P22 nanoreactors.6-9, 12, 14, 32, 41, 43 This truncation

10

results in a protein with molecular weight of 20 kDa (reduced from 33 kDa for SPwt), and

11

an increase in the pI to 9.2 (from 5.2 for SPwt), resulting in a highly positively charged

12

protein at neutral pH. P22 PC capsids, assembled with the truncated SP (P22t), were

13

found to contain 531 ± 3.8 SPt after in vitro assembly and purification, which is much

14

higher than the number of encapsulated proteins observed for full-length wild type SP

15

and also higher than the 420 putative specific binding sites, located in the internal

16

surface of P22, per capsid.31 This suggests that SPt proteins are binding to more sites

17

than just the putative binding sites and are likely binding non-specifically to the P22

18

capsid interior. This is probably due to the high concentration of positive charge of the

19

SPt, which can interact with the net negatively charged interior of P22. Interestingly, the

20

MW of P22t capsids remains constant over a period of 30 days in buffer containing either

21

20 mM, 200 mM, or 1000 mM sodium chloride, indicating SPt do not slowly leak out of

22

the capsid, as is the case for SPwt (Figure 4a; Supp. Figure S9). Another explanation for

23

retention is that SPt are forming oligomers that are too large to traverse the capsid wall.


Page 24 of 36

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Although the original dimerization domain is absent in the SPt, it is possible oligomers

2

can form through a different mechanism. This possibility should be assessed in future

3

work.

4 5

6 7

Figure 4. SPt retention in P22 as a function of time and P22 morphology. a) The number

8

of SPt inside of P22t remains constant over time. b) SDS-PAGE of P22t EX and WB

9

capsids showing retention of a significant portion of SPt.

10 11

To examine whether this retention behavior was due to SPt remaining bound to the

12

putative SP-binding site within the capsid, or bound non-specifically, we expanded the

13

P22t capsids (Supp. Figure S10). Expansion alters the conformation of the CP subunit

14

removing the putative SP binding sites, but the interior of the capsid remains negatively

15

charged.31, 42 P22t PC capsids that were expanded were measured to have a MW of

16

26.2 ± 0.02 MDa, indicating 337 SPt remained bound inside of the capsid after



1

expansion (Supp. Table S3; Figure 4b). During the transition from the PC to the EX

2

morphology the hexameric pores shrink from approximately 2.0 – 2.5 nm to 0.7 – 1.3

3

nm based on PDB structural models (PC:2XYY; EX: 2XYZ, 5UU5). It is unknown if full

4

length SPwt can traverse the pores in the EX structure because it is not known whether

5

they exit the capsid before or after expansion. Therefore, if P22t capsids can expand

6

without releasing SPt, the latter may become trapped after the pores shrink.

7


Page 26 of 36

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

When P22t PCs were converted to WB they were measured to have a MW of 23.3 ± 0.04

2

MDa, suggesting that a significant number of SPt remained encapsulated inside of P22t

3

even in this morphology where large 10 nm pores are created at each of the 12 5-fold

4

axes. The change in MW of SPt capsids from EX to WB was 2.9 MDa, which is similar to

5

the 2.8 MDa mass of the 12 pentamers that are theoretically removed to form WB

6

capsids. While this mass loss may be the result of losing a mixture of SPt and

7

pentamers, it is apparent that a significant number of SPt remain inside of the capsid

8

after the morphological shift, as shown by the presence of band corresponding to SPt on

9

an SDS-PAGE gel. This indicates that SPt are not sterically trapped inside the capsid

10

but instead remain bound to the capsid interior (Figure 4b). Because the MW

11

measurement by MALS cannot distinguish between SP and CP contributions, we

12

cannot say how many SPt remain inside of WB capsids. However, if we take into

13

account the mass loss possible if no pentamers were ejected (which is unlikely because

14

the heated sample shifts on an agarose gel) or if all 12 pentamers were ejected,

15

between 188 and 332 SPt remain inside of P22t WB capsids, the latter value equaling

16

the number of SPt in the EX capsid. Resuspension of either EX of WB capsids in 1 M

17

NaCl buffer resulted in no significant change in MW suggesting CP and SPt may interact

18

through other mechanisms besides electrostatic interactions (e.g. hydrogen bonding

19

and/or hydrophobic interactions). However, treating the EX capsids with 0.5 M GuHCl

20

effectively removed the majority of SPt, resulting in capsids with a MW of 20.6, which is

21

close to the theoretical 19.6 MDa MW of empty EX particles and the same MW as P22

22

capsids that contained SPwt after expansion (Supp. Figure S11). This ability to remove

23

SPt with a chaotrope, but not high ionic strength, suggests that it is necessary to



1

decrease the stability of either the SPt or the CP, or both, to disrupt the binding between

2

the two proteins and allow for SPt removal. Additionally, chaotrope may alter the

3

secondary structure, and thus shape, of the SPt to allow it to traverse capsid wall.

4 5

Conclusion

6

We have shown that the retention of cargo molecules within the P22 VLP is dependent

7

on the nature of both the cargo and the capsid. By altering the ionic strength of the

8

solution, the cargo pI, and the capsid pore size, we have defined a range of conditions

9

in which cargo is preferentially retained or released. The rate of release of the wild type

10

SP can be controlled by altering the ionic strength or temperature, but the majority of

11

the SP eventually exits the capsid. The truncated SP, on the other hand, is retained in

12

the P22 PC morphology, even at higher ionic strengths, over a 30-day period. Because

13

this version of the SP is smaller than the wild type version, and because the pI is much

14

higher, we think that strong electrostatic interactions are causing SPt retention.

15

Interestingly, SPt is also retained in the EX and WB morphologies, even though the

16

putative SP binding site on the capsid interior becomes inaccessible due to

17

conformational changes in CP. This data provides further evidence that SPt is able to

18

bind to other negatively charged regions of the capsid interior. Additionally, cargo

19

fusions to the SPt led to their retention in P22 PC due to size constraints of the cargo

20

and the pores in the PC form. Different results were observed in the WB morphology

21

where GFP-SPt could exit though the WB pores while the CelB-SPt could not and

22

remained encapsulated. We have thus shown that while cargo size is an important


Page 28 of 36

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

consideration for cargo retention inside of the P22 VLP, electrostatic interactions also

2

play an essential role in both the retention of cargo and the rate of cargo release.

3 4

Supporting Information: Additional experimental data.

5 6

Acknowledgements:

7

K.M. and K.S. were supported by a grant from the National Science Foundation (NSF-

8

BMAT DMR-1507282). J.R.C. was funded from the Spanish Ministry of Economy,

9

Industry and Competitivity (BFU2017-88736-R) and the the Comunidad Autónoma de

10

Madrid (S2013/MIT-2807). This research used resources of the Advanced Photon

11

Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for

12

the DOE Office of Science by Argonne National Laboratory under Contract No. DE-

13

AC02-06CH11357. We thank the IU Electron Microscopy Center and the IU Laboratory

14

for Biological Mass Spectrometry for access to their instrumentation.

15 16

References

17

1.

18

protein cages. Nature 1998, 393, (6681), 152-155.

19

2.

20

spectroscopic enhancers for in vitro studies on single viruses. J. Am. Chem. Soc. 2003, 125, (21),

21

6374-6375.

Douglas, T.; Young, M., Host-guest encapsulation of materials by assembled virus

Dragnea, B.; Chen, C.; Kwak, E. S.; Stein, B.; Kao, C. C., Gold nanoparticles as



1

3.

Schwarz, B.; Uchida, M.; Douglas, T., Biomedical and Catalytic Opportunities of Virus-

2

Like Particles in Nanotechnology. In Adv. Virus Res., Vol 97, Kielian, M.; Mettenleiter, T. C.;

3

Roossinck, M. J., Eds. 2017; Vol. 97, pp 1-60.

4

4.

Kilcher, S.; Mercer, J., DNA virus uncoating. Virology 2015, 479, 578-590.

5

5.

Bertin, A.; de Frutos, M.; Letellier, L., Bacteriophage-host interactions leading to genome

6

internalization. Curr. Opin. Microbiol. 2011, 14, (4), 492-496.

7

6.

8

Proteins: Spatially Enforced Communication of GFP and mCherry Encapsulated within the P22

9

Capsid. Biomacromolecules 2012, 13, (12), 3902-3907.

O'Neil, A.; Prevelige, P. E.; Basu, G.; Douglas, T., Coconfinement of Fluorescent

10

7.

O'Neil, A.; Prevelige, P. E.; Douglas, T., Stabilizing viral nano-reactors for nerve-agent

11

degradation. Biomater. Sci. 2013, 1, (8), 881-886.

12

8.

13

sequestration into the P22 VLP. Chem. Commun. 2013, 49, (88), 10412-10414.

14

9.

15

an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle. ACS Chem. Biol. 2014, 9,

16

(2), 359-365.

17

10.

18

M.; LaFrance, B.; Basu, G.; Rynda-Apple, A.; Douglas, T., Symmetry Controlled, Genetic

19

Presentation of Bioactive Proteins on the P22 Virus-like Particle Using an External Decoration

20

Protein. ACS Nano 2015, 9, (9), 9134-9147.

21

11.

22

Uchida, M.; McCoy, K.; Douglas, T., Sortase-Mediated Ligation as a Modular Approach for the

Patterson, D. P.; LaFrance, B.; Douglas, T., Rescuing recombinant proteins by

Patterson, D. P.; Schwarz, B.; Waters, R. S.; Gedeon, T.; Douglas, T., Encapsulation of

Schwarz, B.; Madden, P.; Avera, J.; Gordon, B.; Larson, K.; Miettinen, H. M.; Uchida,

Patterson, D.; Schwarz, B.; Avera, J.; Western, B.; Hicks, M.; Krugler, P.; Terra, M.;


Page 30 of 36

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22 Virus-like Particle.

2

Bioconjugate Chem. 2017, 28, (8), 2114-2124.

3

12.

4

exterior decoration of a virus-like particle. Nanoscale 2017, 9, (29), 10420-10430.

5

13.

6

Assembly of Virus-like Particles (VLPs) Mediated by Multi-valent Protein Linkers. Small 2015,

7

11, (13), 1562-1570.

8

14.

9

LaFrance, B.; Patterson, D.; Schwarz, B.; Karty, J. A.; Prevelige, P. E.; Lee, B.; Douglas, T.,

Sharma, J.; Uchida, M.; Miettinen, H. M.; Douglas, T., Modular interior loading and

Uchida, M.; LaFrance, B.; Broomell, C. C.; Prevelige, P. E.; Douglas, T., Higher Order

Uchida, M.; McCoy, K.; Fukuto, M.; Yang, L.; Yoshimura, H.; Miettinen, H. M.;

10

Modular Self-Assembly of Protein Cage Lattices for Multistep Catalysis. ACS Nano 2017, 12,

11

(2) 942-953.

12

15.

13

Ordered Protein Macromolecular Framework from P22 Virus-like Particles. ACS Nano 2018, 12,

14

(4), 3541-3550.

15

16.

16

Potter, C. S.; Carragher, B.; Johnson, J. E., The structure of an infectious P22 virion shows the

17

signal for headful DNA packaging. Science 2006, 312, (5781), 1791-1795.

18

17.

19

M., Stepwise molecular display utilizing icosahedral and helical complexes of phage coat and

20

decoration proteins in the development of robust nanoscale display vehicles. Biomaterials 2012,

21

33, (22), 5628-5637.

McCoy, K.; Uchida, M.; Lee, B.; Douglas, T., Templated Assembly of a Functional

Lander, G. C.; Tang, L.; Casjens, S. R.; Gilcrease, E. B.; Prevelige, P.; Poliakov, A.;

Parent, K. N.; Deedas, C. T.; Egelman, E. H.; Casjens, S. R.; Baker, T. S.; Teschke, C.



1

18.

Prevelige, P. E.; Fane, B. A., Building the Machines: Scaffolding Protein Functions

2

During Bacteriophage Morphogenesis. In Viral Molecular Machines, Rossmann, M. G.; Rao, V.

3

B., Eds. 2012; Vol. 726, pp 325-350.

4

19.

5

P22 Viral Capsids as Nanoplatforms. Biomacromolecules 2010, 11, (10), 2804-2809.

6

20.

7

Nanoparticle Stability with Symmetrical Morphogenesis. ACS Nano 2016, 10, (9), 8465-8473.

8

21.

9

Structure of the coat protein-binding domain of the scaffolding protein from a double-stranded

Kang, S.; Uchida, M.; O'Neil, A.; Li, R.; Prevelige, P. E.; Douglas, T., Implementation of

Llauro, A.; Schwarz, B.; Koliyatt, R.; de Pablo, P. J.; Douglas, T., Tuning Viral Capsid

Sun, Y. H.; Parker, M. H.; Weigele, P.; Casjens, S.; Prevelige, P. E.; Krishna, N. R.,

10

DNA virus. J. Mol. Biol. 2000, 297, (5), 1195-1202.

11

22.

12

Decoding bacteriophage P22 assembly: Identification of two charged residues in scaffolding

13

protein responsible for coat protein interaction. Virology 2011, 421, (1), 1-11.

14

23.

15

Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat

16

Protein Responsible for Interaction with Scaffolding Protein. J. Virol. 2014, 88, (10), 5287-5297.

17

24.

18

forms oligomers in solution. J. Mol. Biol. 1997, 268, (3), 655-665.

19

25.

20

Scaffolding Protein Induced by Interaction with Coat Protein. J. Mol. Biol. 2011, 410, (2), 226-

21

240.

22

26.

23

in a subunit are not alleviated by assembly. Biochemistry 2000, 39, (5), 1142-1151.

Cortines, J. R.; Weigele, P. R.; Gilcrease, E. B.; Casjens, S. R.; Teschke, C. M.,

Cortines, J. R.; Motwani, T.; Vyas, A. A.; Teschke, C. M., Highly Specific Salt Bridges

Parker, M. H.; Stafford, W. F.; Prevelige, P. E., Bacteriophage P22 scaffolding protein

Padilla-Meier, G. P.; Teschke, C. M., Conformational Changes in Bacteriophage P22

Capen, C. M.; Teschke, C. M., Folding defects caused by single amino acid substitutions


Page 32 of 36

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

27.

Fuller, M. T.; King, J., Purification of the Coat and Scaffolding Proteins from Procapsids

2

of Bacteriophage P22. Virology 1981, 112, (2), 529-547.

3

28.

4

Virology 1994, 205, (1), 188-197.

5

29.

6

two distinct scaffolding protein binding populations within the bacteriophage P22 procapsid.

7

Biochemistry 2001, 40, (30), 8962-8970.

8

30.

9

scaffolding protein. J. Mol. Biol. 1998, 281, (1), 69-79.

Greene, B.; King, J., Binding of scaffolding subunts within the P22 procapsid lattice.

Parker, M. H.; Brouillette, C. G.; Prevelige, P. E., Kinetic and calorimetric evidence for

Parker, M. H.; Casjens, S.; Prevelige, P. E., Functional domains of bacteriophage P22

10

31.

Chen, D. H.; Baker, M. L.; Hryc, C. F.; DiMaio, F.; Jakana, J.; Wu, W. M.; Dougherty,

11

M.; Haase-Pettingell, C.; Schmid, M. F.; Jiang, W.; Baker, D.; King, J. A.; Chiu, W., Structural

12

basis for scaffolding-mediated assembly and maturation of a dsDNA virus. Proc. Natl. Acad. Sci.

13

U. S. A. 2011, 108, (4), 1355-1360.

14

32.

15

Douglas, T., Virus-like particle nanoreactors: programmed encapsulation of the thermostable

16

CelB glycosidase inside the P22 capsid. Soft Matter 2012, 8, (39), 10158-10166.

17

33.

18

of P22 coat subunits into icosahedral shells in vitro. J. Mol. Biol. 1988, 202, (4), 743-757.

19

34.

20

T., Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical

21

polymerization with high-density cargo loading. Nat. Chem. 2012, 4, (10), 781-788.

Patterson, D. P.; Schwarz, B.; El-Boubbou, K.; van der Oost, J.; Prevelige, P. E.;

Prevelige, P. E.; Thomas, D.; King, J., Scaffolding protein regulates the polymerization

Lucon, J.; Qazi, S.; Uchida, M.; Bedwell, G. J.; LaFrance, B.; Prevelige, P. E.; Douglas,



1

35.

Marabini, R.; Masegosa, I. M.; SanMartin, M. C.; Marco, S.; Fernandez, J. J.; delaFraga,

2

L. G.; Vaquerizo, C.; Carazo, J. M., Xmipp: An image processing package for electron

3

microscopy. J. Struct. Biol. 1996, 116, (1), 237-240.

4

36.

5

C.; Ferrin, T. E., UCSF Chimera--a visualization system for exploratory research and analysis. J.

6

Comput. Chem. 2004, 25, (13), 1605-12.

7

37.

8

in electron microscopy. J. Struct. Biol. 2003, 142, (3), 334-347.

9

38.

Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E.

Mindell, J. A.; Grigorieff, N., Accurate determination of local defocus and specimen tilt

Sorzano, C. O.; Bilbao-Castro, J. R.; Shkolnisky, Y.; Alcorlo, M.; Melero, R.; Caffarena-

10

Fernandez, G.; Li, M.; Xu, G.; Marabini, R.; Carazo, J. M., A clustering approach to

11

multireference alignment of single-particle projections in electron microscopy. J. Struct. Biol.

12

2010, 171, (2), 197-206.

13

39.

14

Jiang, W.; Adams, P. D.; King, J. A.; Schmid, M. F.; Chiu, W., Accurate model annotation of a

15

near-atomic resolution cryo-EM map. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, (12), 3103-3108.

16

40.

17

Image processing for electron microscopy single-particle analysis using XMIPP. Nat. Protoc.

18

2008, 3, (6), 977-990.

19

41.

20

Programmed In Vivo Packaging of Protein Cargo and Its Controlled Release from Bacteriophage

21

P22. Angew. Chem., Int. Ed. 2011, 50, (32), 7425-7428.

Hryc, C. F.; Chen, D. H.; Afonine, P. V.; Jakana, J.; Wang, Z.; Haase-Pettingell, C.;

Scheres, S. H. W.; Nunez-Ramirez, R.; Sorzano, C. O. S.; Carazo, J. M.; Marabini, R.,

O'Neil, A.; Reichhardt, C.; Johnson, B.; Prevelige, P. E.; Douglas, T., Genetically


Page 34 of 36

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

42.

Llauro, A.; Luque, D.; Edwards, E.; Trus, B. L.; Avera, J.; Reguera, D.; Douglas, T.; de

2

Pablo, P. J.; Caston, J. R., Cargo-shell and cargo-cargo couplings govern the mechanics of

3

artificially loaded virus-derived cages. Nanoscale 2016, 8, (17), 9328-9336.

4

43.

5

nanoparticles by encapsulation of hydrogen peroxide producing enzyme inside the P22 VLP. J.

6

Mater. Chem. B 2014, 2, (36), 5948-5951.

Patterson, D. P.; McCoy, K.; Fijen, C.; Douglas, T., Constructing catalytic antimicrobial

7 8 9 10 11 12 13 14 15 16 17 18

TOC Graphic

19 20



1 2


Page 36 of 36

Cargo Retention inside P22 Virus-Like Particles

Recommend Documents