A synthetic polymer affinity ligand for Bacillus thuringiensis (Bt

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A synthetic polymer affinity ligand for Bacillus thuringiensis (Bt) Cry1Ab/Ac protein. The use of biomimicry based on the Bt protein-insect receptor binding mechanism. Mingming Liu, Rong Huang, Adam C Weisman, Xiaoyang Yu, Shih-Hui Lee, Yalu Chen, Chao Huang, Senhua Hu, Xiuhua Chen, Wenfeng Tan, Fan Liu, Hao Chen, and Kenneth J. Shea J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b01710 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Journal of the American Chemical Society

1

A synthetic polymer affinity ligand for Bacillus thuringiensis (Bt)

2

Cry1Ab/Ac protein. The use of biomimicry based on the Bt

3

protein-insect receptor binding mechanism.

4

5

Mingming Liua,c*, Rong Huanga, Adam Weismanc, Xiaoyang Yua, Shih-Hui Leec,

6

Yalu Chena, Chao Huanga, Senhua Hua, Xiuhua Chena,

7

Wenfeng Tana, Fan Liua, Hao Chenb, Kenneth J. Sheac*

8

a

9

Yangtse River), Ministry of Agriculture, College of Resources and Environment,

Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of

10

Huazhong Agricultural University, Wuhan 430070, China.

11

b

College of Science, Huazhong Agricultural University, Wuhan 430070, China.

12

c

Department of Chemistry, University of California-Irvine, Irvine, California 92697,

13

United States

14

* Corresponding authors.

15

Tel.: +86 27 8728 0271. Fax: +86 27 8728 8618.

16

E-mail address: [email protected] (M.-m. Liu); [email protected] (K.J. Shea).

17 18 19 20 21 22

1

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Journal of the American Chemical Society 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

ABSTRACT

2

We report a novel strategy for creating abiotic Bacillus thuringiensis (Bt) protein

3

affinity ligands by biomimicry of the recognition process that takes place between Bt

4

Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R1 proteins. Guided by this

5

strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and

6

screened for binding to three epitopes

7

and 436FRSGFSNSSVSIIR449 located in loop α8, loop 2 and loop 3 of domain II of Bt

8

Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was

9

found to have high affinity to one of the epitopes, 368RRPFNIGINNQQ379. This same

10

NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that

11

share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F

12

closely related proteins that lack epitope homology. To locate possible NP-Bt

13

Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the

14

binding of 865NITIHITDTNNK876, a specific recognition site in insect receptor Bt-R1,

15

to 368RRPFNIGINNQQ379. The inhibition by NP12 reached as high as 84%, indicating

16

that NP12 binds to Bt Cry1Ab/Ac proteins mainly via

17

epitope region was then utilized as a “target” or “bait” for the separation and

18

concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by

19

NP12. This strategy, based on the antigen-receptor recognition mechanism, can be

20

extended to other biotoxins and pathogen proteins when designing biomimic

21

alternatives to natural protein affinity ligands.

280

FRGSAQGIEGS290, 368RRPFNIGINNQQ379

22 23 2


368

RRPFNIGINNQQ379. This

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1

n INTRODUCTION

2

Bacillus thuringiensis (Bt) is an aerobic, spore-forming bacteria that produces

3

crystalline inclusions called insecticidal crystal proteins (ICPs) during the sporulation

4

phase.1 There are thousands of individual ICPs discovered to date, of which the

5

majority belong to the Cry or Cyt toxin family.2 These crystalline proteins are

6

specifically toxic to larvae of several insect orders, including Lepidoptera, Coleoptera,

7

Hymenoptera, Diptera and Nematodes, some of which are pests of agricultural plants.

8

Therefore, Bt has been used worldwide in biological control of crop pests in

9

agriculture, either as spray formulations or as a source of genes encoding insecticidal

10

proteins for transgenic crops.3 However, Bt proteins released from root exudates and

11

plant residues can accumulate in aqueous and soil ecosystem and retain their

12

larvicidal activity for a long time, which may pose a potential threat to non-target

13

species and ultimately lead to unexpected ecosystem-scale consequences.4,5 In general,

14

Bt proteins present in soil and transgenic Bt crop samples are found at concentrations

15

as low as ng/g to sub µg/g range.6,7 A variety of analytical methods have been

16

employed for the detection of low-abundant Bt proteins in transgenic crop tissues and

17

residues, water and soil samples. These include bioassays,8 enzyme-linked

18

immunosorbent assay (ELISA),7 lateral flow immunoassay,9 Western blot,10

19

protein-based Immuno-PCR11 and size exclusion high performance liquid

20

chromatography (HPLC)12. A major bottleneck of these methods, however, is the lack

21

of efficient extraction and separation strategies for the low-abundant Bt proteins. At

22

present, an ELISA assay following liquid-liquid extraction is still the most common

3



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1

detection method for Bt proteins. The buffer systems used for liquid-liquid extraction

2

include phosphate-buffered saline with Tween 20, sodium carbonate buffer, sodium

3

dodecyl sulfate and an invertebrate gut fluid solution.7,13,14 The poor extraction and

4

separation efficiency of these methods frequently leads to detection errors and

5

frequent false-negative results. A rapid, sensitive and selective separation method for

6

low abundant Bt proteins in environmental and transgenic Bt crop samples would be a

7

valuable addition to the analytical repertoire.

8

Synthetic polymer nanoparticles (NPs) with the capacity to selectively bind

9

target biomacromolecules are of significant interest as abiotic alternatives to

10

biological affinity ligands, such as antibodies and protein receptors, for applications in

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protein isolation/purification,15-21 toxicity neutralization,22-28 disease diagnostics and

12

therapeutics29-32. In nature, antibodies and protein receptors recognize antigens by a

13

combination of multiple weak interactions, including electrostatic, hydrogen-bonding,

14

van der Waals, hydrophobic, and/or π-π stacking interactions on the complementary

15

protein interface. To mimic these interactions, polymer NPs have been developed with

16

high biomacromolecule affinity by screening for optimized combinations and ratios of

17

monomers with charged, hydrophilic, hydrophobic or aromatic functional groups

18

complementary

19

biomacromolecules.17,22,25,33-36 These studies focused on the synthesis and screening

20

of polymer NPs with high affinity to biogenic targets including peptides,22,25

21

proteins,15,17 carbohydrates,37 viruses,38 bacteria39 and cells40. However, many of these

22

studies were based on a trial-and-error screening approach, which can be tedious,

to

exposed

amino

acid

residues

4


of

the

target

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time-consuming and not always resulting in NPs with optimal affinity and specificity.

2

Therefore, discovery of more efficient and effective strategies for creating synthetic

3

protein affinity nanoparticles is of particular importance. Targeting epitopes, an

4

approach used in antibody selection41 and molecular imprinting,42-45 is one such

5

direction that offers a more focused approach. We report here a directed chemical

6

evolution21 of a synthetic polymer hydrogel with affinity for epitopes of Bt Cry

7

proteins. This directed chemical evolution is based upon the recognition process that

8

takes place between Bt proteins and insect receptors, with the hope that it could

9

provide abiotic Bt protein affinity ligands (NPs) that mimic the cadherin-like protein

10

insect receptors.

11

The mechanism of insecticidal activity of Bt proteins is complex. The Bt Cry

12

proteins, which commonly exist as inactive protoxins, when ingested by susceptible

13

insects are solubilized in the alkaline midgut and proteolytically cleaved by midgut

14

proteases to produce active toxins with molecular weights that range from 60-70

15

KDa.46,47 The activated Bt Cry toxins are composed of three conserved domains

16

(Figure 1a).1,46,48,49 Domain I contains a seven α-helix bundle and is involved in

17

oligomer formation, membrane insertion and pore formation. Domain II is a β-prism

18

that consists of three antiparallel β-sheets with exposed loop regions and is implicated

19

in receptor interaction and insect specificity.50 Domain III is a β-sandwich that is

20

composed of two antiparallel β-sheets and is also involved in receptor interaction,

21

insect specificity and stabilization of molecular structure. 51 The binding of

5



1

2

Figure 1. (a) The structure of Bt Cry1Ac proteins. The PDB ID is 4ARX, and the UniProtKB AC

3

is P05068. (b) The structure model of the insect receptor cadherin-like protein Bt-R1.

4 5

activated Bt Cry toxins to the specific receptors located on the brush border membrane

6

of midgut epithelial cells, has proved to be a critical step in the toxic pathway of Bt

7

Cry proteins to susceptible insect species.52,53 These receptors in the insect midgut

8

include the glycosylphosphatidylinositol-anchored proteins, known as alkaline

9

phosphatase54,55 or aminopeptidase N,56,57 and the cadherin-like proteins, known as

10

Bt-R158,59 and BtR17557-62. Among them, the cadherins, which are composed of an

11

aminoterminal signal sequence, an ectodomain formed by 11 or 12 cadherin repeats

12

(CRs), a membrane-proximal extracellular domain, a transmembrane domain and a

13

cytoplasmic domain, are the most thoroughly studied receptors (Figure 1b).63 Many

14

studies have indicated that the insecticidal Bt Cry proteins can bind to the 6


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1

cadherin-like receptors in Manduca sexta, Heliothis virescens, and Bombyx mori via

2

the surface exposed charged and hydrophobic residues in loop α8,51 loop 2,64 and loop

3

3 of domain II of Bt Cry proteins65. For example, the loop 2

4

and loop α8 280FRGSAQGIEGS290 in domain II of Bt Cry1Ab protein has been shown

5

to bind with the sequence

6

CR7 and CR11 of the Manduca sexta receptor cadherin-like protein Bt-R1,51,58 and the

7

loop 3

8

shown to interact with the sequence

9

Heliothis virescens cadherin-like receptor59,65.

436

865

NITIHITDTNNK876,

1331

368

RRPFNIGINNQQ379

IPLPASILTVTV1342 located in

FRSGFSNSSVSIIR449 in domain II of Bt Cry1Ab/Ac protein has been 1423

GVLTLNFQ1431 located in CR12 of the

10

Motivated by these studies, we propose a new strategy to create synthetic Bt

11

protein affinity reagents by biomimicry of the recognition process that takes place

12

between Bt Cry1Ab/Ac protein and the cadherin-like protein Bt-R1 insect receptor

13

(Figure 2). This strategy takes cognizance of the fact that a synthetic polymer NP can

14

“map” onto a specific protein or peptide surface via multiple weak interactions

15

complementary to exposed amino acid residues in this region of the target protein.

16

The target of the synthetic polymer NP is analogous to that of the natural biological

17

receptors. Bt Cry1Ab/Ac proteins bind to the cadherin-like protein Bt-R1 insect

18

receptor via three specific peptide epitopes on the Bt Cry1Ab/Ac proteins,

19

280

20

pI 12.0) or

21

to develop synthetic polymer NPs that are engineered with intrinsic affinity to one or

22

more of these three epitopes of Bt Cry1Ab/Ac proteins. The abiotic receptor selected

FRGSAQGIEGS290 (MW 1108.18, pI 6.0), 436

368

RRPFNIGINNQQ379 (MW 1456.64,

FRSGFSNSSVSIIR449 (MW 1556.76, pI 12.0).51,58,59,65 Our approach is

7



Page 8 of 41

1

by this strategy would mimic the insect cadherin Bt-R1 receptor and “target” or “bait”

2

the epitope region of Bt Cry1Ab/Ac proteins. Realization of this approach would

3

enable recognition, capture and separation of Bt Cry1Ab/Ac proteins from complex

4

biological and environmental matrices with a high-affinity abiotic NP receptor.

5 6 7 Binding site: epitope

8 9 10 Insect receptor cadherinlike protein Bt-R1

11 Bt Cry1Ab/Ac

12

Bt Cry1Ab/Ac―Bt-R1 binding

13 14 15

Binding site: epitope?

16 Polymer NP

17 18 19

Bt Cry1Ab/Ac

Bt Cry1Ab/Ac—polymer NP binding

20 21

Figure 2. The sketch of the biomimicry strategy to create synthetic Bt protein affinity

22

nanoparticles based on Bt Cry1Ab/Ac protein-insect receptor cadherin-like protein Bt-R1 binding

23

mechanism.

8


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1

Guided by this strategy, a library of synthetic polymer NPs incorporating various

2

charged, hydrophilic and hydrophobic groups were synthesized and then screened for

3

affinity against the three peptide epitopes, and the corresponding Bt Cry1Ab protein.

4

A NP with epitope and Bt Cry1Ab protein affinity was selected for subsequent studies.

5

Biomimicry of the NP was confirmed by a competition experiment using the NP as

6

inhibitor to block the binding of

7

Bt-R1 insect receptor to the high-affinity epitope in domain II of Bt Cry1Ab protein.

8

NP selectivity was also evaluated by comparing its affinity to Bt Cry1Ab (65

9

KDa, >85 %) and Cry1Ac (64 KDa, >85 %) proteins that share the same epitope, and

10

Bt Cry2A (68 KDa, >95 %), Cry1C (67 KDa, >90 %) and Cry1F (68 KDa, >95 %)

11

proteins that lack epitope homology. Finally, the utility of the engineered NP for

12

sequestering Bt Cry1Ac proteins from the extract of transgenic Bt cotton leaves was

13

demonstrated.

865

NITIHITDTNNK876, a recognition domain in the

14 15

n RESULTS AND DISCUSSION

16

Nanoparticle Library. A library of synthetic polymer NPs was prepared

17

by precipitation polymerization. The polymers were comprised of combinations and

18

ratios of functional monomers that included hydrophilic acrylamide (AAm),

19

N-isopropylacrylamide (NIPAm), negatively charged acrylic acid (AAc), positively

20

charged (3-acrylamidopropyl)trimethylammonium chloride (ATC),

21

N-(3-aminopropyl)methacrylamide hydrochloride (APM), and hydrophobic

22

N-tert-butylacrylamide (TBAm). Table 1 summarizes the monomer feed ratio,

9



Page 10 of 41

1

Table 1 The monomer feed ratios and characterization parameters of the synthetic polymer

2

nanoparticles. NPs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

3

TBAm 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40

Monomer feed ratio (mol %) NIPAm AAm AAc APM 58 0 53 5 48 10 38 20 28 30 18 40 58 0 53 5 48 10 38 20 28 30 18 40 0 58 58 0 53 5 48 10 38 20 53 48 38 28 18 0 -

ATC 5 10 20 30 40 58

Bis 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Diameter (nm) 74 122 157 214 275 270 274 3513 4016 2609 236 175 219 aggregation 37 38 59 26 44 54 2262 695 732

PDI 0.057 0.127 0.057 0.040 0.013 0.039 0.772 1.000 1.000 1.000 0.003 0.007 0.103 0.265 0.391 0.511 0.379 0.700 0.708 1.000 0.730 0.810

Concentration (mg/mL) 5.04 5.68 5.60 5.10 5.22 5.44 4.24 4.34 3.96 3.82 3.70 4.10 4.10 3.82 4.60 4.00 3.60 5.42 4.58 3.78 5.32 5.27 5.80

% yield 65 92 91 90 76 94 75 80 75 68 69 74 74 74 68 66 74 71 60 58 57 53 53

4 5

hydrodynamic diameter, yield and polymer concentration of the synthetic NPs. All

6

NPs, except NP14, showed relatively high colloidal stability and narrow size

7

distribution in water.

8

Binding Study between Synthetic Polymer NPs and Epitopes of

9

Bt Cry1Ab/Ac Proteins. The binding capacity of the three epitopes

10

280

FRGSAQGIEGS290,

368

RRPFNIGINNQQ379 and

436

FRSGFSNSSVSIIR449 on NPs

11

1-23 was evaluated by preincubating the peptides (40 µg/mL) and NPs (0.5 mg/mL)

12

in 35 mM phosphate buffer (pH 7.43). The results are summarized in Figure 3. The

13

capture efficiencies for 280FRGSAQGIEGS290 and 436FRSGFSNSSVSIIR449 were very

10


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23 N P P 22 1 N P2 0 N P2 9 N P1 8 N P1 N P 17 N 16 N P P 15 4 N 1 N P 13 N P P 12 1 N P1 0 N P1 9 N NP 8 NP 7 NP P 6 N 5 NP 4 NP

1

10 5 0 368

436

NP

3 NP

2 NP

280

1

s ( m

R R P F NIG INNQ Q

F R S G F S NS S V S IIR

F R G S A Q G IE G S

r p ti o

15

n a m

20

ount

25

g /g )

30

A ds o

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


379

449

290

2

Figure 3. Comparison of the binding capacity of the peptide epitope

3

368

4

mM pH 7.43 phosphate buffer.

280

FRGSAQGIEGS290,

RRPFNIGINNQQ379 and 436FRSGFSNSSVSIIR449 (40 µg/mL) on NPs 1-23 (0.5 mg/mL) in 35

5 368

RRPFNIGINNQQ379

6

low for all NPs studied. However, significant amounts of

7

were adsorbed by negatively charged AAm- and NIPAm-based NPs 1-13; positively

8

charged APM- and ATC-based NPs 14-23 on the other hand showed little affinity for

9

this peptide. That the negatively charged NPs 1-13 showed greater binding to

10

368

RRPFNIGINNQQ379 (positively charged at pH 7.43) than to the negatively charged

11

280

FRGSAQGIEGS290 is likely due to contributions to binding from electrostatic

12

interactions.

13

368

14

both peptides contain two positive arginine residues and possess similar isoelectric

15

points (pI 12.0). It is clear therefore that electrostatics are not the only factor

Interestingly,

the

binding

capacities

RRPFNIGINNQQ379 were higher than that of

436

NPs

1-13

for

FRSGFSNSSVSIIR449 although

11

of



Page 12 of 41

1

contributing to NP-epitope binding. This difference likely arises from differences in

2

both hydrophilic and hydrophobic amino acid residues between these two peptides

3

suggesting that hydropathic complementarity is a critical determinant of peptide

4

binding to NPs 1-13. The preceding findings led to the selection of epitope

5

368

6

in subsequent studies.

RRPFNIGINNQQ379 as the “target” or “bait” for the capture of Bt Cry1Ab protein

For

7

the

negatively

charged

NPs

1-13,

the

binding

capacity

of

8

368

RRPFNIGINNQQ379 increased with an increase in the AAc feed ratio (Figure 3

9

and Figure S1). There were also differences in the binding capacity of

10

368

RRPFNIGINNQQ379 between NIPAm and AAm-based NPs. The increase was

11

more pronounced for the AAm-based NPs 9-12 than that for the NIPAm-based NPs

12

3-6 as the feed ratio of AAc increased from 10 to 40%. Significant differences

13

between AAm- and NIPAm-based NPs in their interactions with biological

14

macromolecules have previously been noted. In a related study, a comparison of the

15

thermodynamics of binding of NIPAm- and AAm-based NPs with similar loadings of

16

identical positively charged monomers to a polyanionic biomacromolecule (heparin)

17

revealed hydrogen bonding played an important role in AAm-based NPs, but not in

18

NIPAm-based NPs.66 Moreover, the AAm-based polymers have lower hydrophobicity

19

and steric hindrance to present to the protein interface compared to the NIPAm-based

20

polymers. However just as protein-protein interactions involve multiple weak

21

interactions care must be exercised to attribute NP binding to a single dominant factor.

22

To

support

this

viewpoint,

we

prepared

two

12


mutated

peptides

of

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1

368

RRPFNIGINNQQ379. The mutant

2

pI 12.0) was prepared by substituting the four hydrophilic amino acid residues NNQQ

3

with non-hydrophilic residues AAAA, and the mutant

4

1286.41, 90.13 %, pI 5.57) was prepared by replacing the two positively charged RR

5

amino acid residues by uncharged AA ones. NP12 uptake of peptide

6

368

RRPFNIGINNQQ379

7

368

AAPFNIGINNQQ379.is given in Figure S2. The result shows that the

8

NP12-peptide affinity decreased significantly by substitution of either hydrophilic or

9

positively charged amino acid residues with non-hydrophilic or uncharged ones,

10

indicating a positive synergistic effect of both electrostatic and hydrogen bonding

11

interactions on

12

AAc, 18% AAm) with high feed ratios of hydrophobic, negatively charged and

13

hydrophilic monomers was used in subsequent studies.

368

and

its

368

RRPFNIGIAAAA379 (MW 1256.47, 95.98 %,

two

mutants

368

368

AAPFNIGINNQQ379 (MW

RRPFNIGIAAAA379

and

RRPFNIGINNQQ379 binding to NP12. This NP (40% TBAm, 40%

14

Besides the monomer composition, the conditions used to evaluate protein

15

binding also play a critical role in the observed NP-biomacromolecule interaction.

16

Figure 4 summarizes the influence of pH, phosphate and salt concentration on the

17

NP12-368RRPFNIGINNQQ379 interaction. This study provides further insight to the

18

contributions of electrostatic/hydrogen bonding/hydrophobic interactions to

19

NP12-368RRPFNIGINNQQ379 binding. In 35 mM phosphate buffer, an increase in pH

20

from 5.26 to 6.20 resulted in a 2.1-fold increase in peptide uptake, while a further

21

increase in pH value from 6.20 to 7.76 resulted in a 2.9-fold decrease in binding

22

capacity of

368

RRPFNIGINNQQ379 . An optimal pH for binding two charged

13


Journal of the American Chemical Society 368

R R P F NIG INNQ Q

379

50

40

30

20

l

l M

B

B

S

S

+1

50

m

m

N

N M

M m

0 +3

aC

aC

B P

P M

m

5 3

76

P M

m 10

pH

6

.0

9

9

10

m

M

P

pH

7.

.1 7

.8 5 pH

1

S

S

S

B

B

P M m

8

35

35

m

0

6

.2

pH

6 .2

pH

5

B

S

S M

M m

35

20 0

.1 7

P

P

P M

m

M m

20 5

.0

pH

6

B

B

S

S

P

P M

m

20 7

5

.1

pH

B

B

S

S

B P M pH

7

.2

6

10

10

m

m

3 6

.9

pH

M

10 pH

6

.0

5

m

M

P

P

B

B

S

S B P

M m 2

10 5

.9 4

pH

pH

6

.0

8

5 .6 4

pH

pH

4

.5

7

2

0m

m

M

M

P

P

B

B

S

S

0

S

10

pH

A d s o rp tio n a m o u n ts ( m g /g )

60

pH

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

Page 14 of 41

2

Figure 4. The influence of pH, phosphate buffer and salt concentration on NP12 (0.5

3

mg/mL)-368RRPFNIGINNQQ379 (40 µg/mL) interaction.

4 5

macromolecules is frequently observed. This is due in part to the fact that the acid

6

dissociation constant (pKa) of ionizable groups on synthetic polymers or amino acid

7

side chains of proteins are often different from those of the isolated functional groups

8

in solution. This arises as a result of interferences by electrostatic interactions such as

9

salt bridges, electrostatic repulsion and hydrogen bonding, hydrophobic interactions

10

and/or van der Waals interactions with other residues and/or amide back bones of

11

poly-peptides.67-70 The interactions between polyfunctional macromolecules often

12

challenge a simple interpretation of changes in a single variable. The binding capacity

13

on NP12 for

14

approximately 6.0. All one can conclude is that electrostatic interactions play an

15

important but not exclusive role in NP12-368RRPFNIGINNQQ379 binding under these

368

RRPFNIGINNQQ379 was highest when the pH value was

14


Page 15 of 41 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

conditions. Further support for this comes from the study of the influence of

2

phosphate

3

368

4

bound to NP12 decreased as the phosphate concentration increased from 2 mM to 35

5

mM, and salt concentration increased from 0 mM to 150 mM at pH 6.0. An increase

6

in salt concentration results in an attenuation of electrostatic interactions71 supporting

7

the

8

NP12-368RRPFNIGINNQQ379 binding. In addition to electrostatic interactions,

9

hydrogen bonding and hydrophobic interactions are also likely contributors to the

and

salt

concentration

on

affinity

RRPFNIGINNQQ379. It was found that the amount of

suggestion

that

electrostatic

368

interactions

between 368

NP12

and

RRPFNIGINNQQ379

play

a

role

in

RRPFNIGINNQQ379. This is supported by

10

overall affinity between NP12 and

11

investigating affinity at low pH (e.g. 4.57 and 4.85) where most carboxylate groups

12

are not dissociated. The binding capacity of

13

pronounced at the two pH values studied. The affinity therefore between NP12 and

14

368

15

and hydrophobic interactions (hydropathic complementarity) all of which can be

16

influenced by binding conditions. From these studies the optimal conditions for

17

368

18

added NaCl.

368

RRPFNIGINNQQ379 is still

RRPFNIGINNQQ379 arises from a combination of electrostatic/hydrogen bonding

RRPFNIGINNQQ379 binding to NP12 is 2 mM phosphate buffer (pH 6.0) without

19

Besides the multiple weak interactions, the local peptide structure may be

20

another important factor affecting the NP12-368RRPFNIGINNQQ379 binding. The

21

possibility that the peptide structure may or may not change with buffer conditions

22

was first considered. Figure S3 gives the circular dichroism (CD) spectra of

15



Page 16 of 41

1

368

RRPFNIGINNQQ379.. The spectra has the features of a random coil. The positions

2

and intensities of the peaks are independent of pH, PBS and NaCl concentration

3

indicating

4

368

that

the

of

these

variables

on

the

conformation

of

RRPFNIGINNQQ379 is very limited. The spectra of

5

impact

368

RRPFNIGINNQQ379 in the presence of NP12 is shown in

6

Figure S3. The CD spectra now exhibits a maximum at (-) 220nm suggesting a

7

structural change upon association with NP12. The CD curve does not strictly

8

conform to any of the standard sub structural motifs. It may imply a composite of

9

species with β-turns and random coils. Some caution should be exercised with

10

assuming that the origin of the CD shift arises exclusively from the peptide-NP

11

interaction since it is quite likely that the NP takes up many copies of the peptide.

12

Spectral changes could result in that case from peptide-peptide interactions within the

13

NP. Despite possible peptide conformational changes upon binding, there should be

14

little difference in NP binding to this epitope in Bt Cry1Ab/Ac since the epitope

15

368

16

domain is typically regarded as a random coil (Figure 1a). The affinity of NP12 to the

17

“free” peptide

18

protein via the domain II loop 2 region of these proteins should be quite similar

19

regardless of whether free or

RRPFNIGINNQQ379 is found in a large loop domain of Bt Cry1Ab/Ac. This

368

RRPFNIGINNQQ379 or its potential to capture Bt Cry1Ab/Ac

“attached” as a part of Bt Cry1Ab/Ac protein.

20

Binding Study between Synthetic Polymer NPs and the Bt

21

Cry1Ab Proteins. A binding study similar to the preceding using the library of

22

synthetic polymer nanoparticles (NP1-23) against Bt Cry1Ab proteins was

16


Page 17 of 41 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

subsequently carried out. The results are summarized in Figure S4. Bt Cry1Ab

2

proteins contain the three epitopes 368RRPFNIGINNQQ379, 280FRGSAQGIEGS290 and

3

436

4

similar pattern of NP-Bt Cry1Ab protein affinity was found. The highest binding

5

capacity for the Bt Cry1Ab protein was observed for NP12 (40% AAc, 18% AAm)

6

(Figure S4), the same NP that binds strongly to the epitope

7

(Figure

8

368

9

and support our approach of using the high affinity epitope as a “target” or “bait” for

10

FRSGFSNSSVSIIR449 that were screened in the preceding study. Importantly, a

3

and

S1).

These

results

strongly

368

RRPFNIGINNQQ379

suggest

that

the

epitope

RRPFNIGINNQQ379 is the putative binding site of the NP12-Bt Cry1Ab interaction

the capture of Bt Cry1Ab protein by NP12.

11

From a study of the influence of pH and ionic strength on Bt Cry1Ab protein

12

binding to NP12 (Figure S5) it was found that the amount of protein adsorbed

13

decreased significantly with an increase in pH (6.05-7.26) and salt concentration

14

(0-150 mM) under the same phosphate concentration (10 mM). Moreover, an increase

15

in phosphate concentration (2-35 mM, pH~6.0) also led to a decrease in binding

16

capacity of Bt Cry1Ab. The amount of Bt Cry1Ab bound to NP12 was highest when

17

the binding was performed in 2 mM pH 6.0 phosphate buffer without NaCl. These

18

conditions are very similar to that found for NP12 binding to the epitope

19

368

RRPFNIGINNQQ379 (Figure 4), a finding that also suggests the epitope

20

368

RRPFNIGINNQQ379 is the nexus of the NP12-Bt Cry1Ab interaction.

Inhibition of 865NITIHITDTNNK876 Binding to 368RRPFNIGINNQQ379

21 22

by NP12. A number of studies indicated that the peptide 17


865

NITIHITDTNNK876


Page 18 of 41

1

located in CR7 of the cadherin-like protein Bt-R1 insect receptor binds specifically to

2

the epitope

3

protein.51,58 To help identify the location of the dominant NP12-Bt Cry1Ab/Ac

4

interaction, a competitive inhibition experiment was carried out in which the NP12

5

was used as an inhibitor to block the binding of

6

368

7

NP12 was first incubated in 10 mM phosphate buffer solution until a binding

8

equilibrium was reached. The peptide

9

buffer solution was then added and incubated to reach a second binding equilibrium.

10

A control experiment was also performed according to the same procedure except that

11

NP12 was not added to the incubation system. Following the binding experiment, the

12

resulting solution was filtrated through a MWCO 100 KDa omega membrane in a

13

centrifugal filter. Peptide uptake was corrected for nonspecific binding. Details of the

14

experiments can be found in the supporting information. The residual concentration of

15

865

16

control group if NP12 and

17

(368RRPFNIGINNQQ379). Otherwise, the residual concentration of

18

865

19

control group if binding of

20

865

368

RRPFNIGINNQQ379 located in domain II loop 2 of Bt Cry1Ab/Ac

RRPFNIGINNQQ379 (Figure 5a). The solution of

865

368

NITIHITDTNNK 876 to

RRPFNIGINNQQ379 and

NITIHITDTNNK876 dissolved in the same

NITIHITDTNNK876 in the experimental group will be higher than that in the 865

NITIHITDTNNK876 both compete for the same peptide

NITIHITDTNNK876 in the experimental group will be the same as that in the 368

RRPFNIGINNQQ379 on NP12 differs from that on

NITIHITDTNNK876. The inhibition of the binding of

21 22

18

865


865

NITIHITDTNNK876 to

Page 19 of 41

2.5

1

p H 6.0 p H 9.3 p H 12.5

2.0

2

ΔA

1.5

1.0

3

0.5

4

(c )

0.0

0 2 :2 0 4 :2 0 6 :2 0 8 :2 0 1 0 :2 0 1 2 :2 0 1 4 :2 0 1 6 :2 0 1 8 :2 0 2 0 :2 0 2 2 :2 0

368

5

R R P F NIG INNQ Q

379 865 876 / NIT IH IT D T NNK

6 ( µg /m L )

200

P u re c o n tro ls Mix ed s am p les 150

865

8

10

368

40

(b )

0

17

8.0

6.0

10.0

865

( µg /m L )/ NIT IH IT D T NNK

160/160 876

( µg /m L )

C ontrol g roup E x perim ental g roup

0.8

0.6

84%

63% 0.4

82%

69% 0.2

60%

(e)

0.0 80/80

12.0

80/160

80/120

120/120

160/160

865 368 379 876 R R P F NIG INNQ Q ( µg /m L )/ NIT IH IT D T NNK ( µg /m L )

pH

18

R R P F N IG IN N Q Q 379 (w t/w t, m g /m g )

16

80

379

120/120

368

14 15

120

865

13

160

A m o u n t o f

12

1.0

379 R R P F NIG INNQ Q ( 80 µg /m L ) 865 876 NIT IH IT D T NNK ( 80 µg /m L )

80/160

80/120

R R P F NIG INNQ Q

368

N IT IH IT D T N N K 876 b o u n d to

200

(d )

0 80/80

11

50

R e s id u a l

9

100

N IT IH IT D T N N K

8 7 6

7

A d s o rp tio n c a p a c ity ( µg /g )

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


865

NITIHITDTNNK876 to

19

Figure 5. (a) Schematic of the inhibition of the binding of

20

368

21

inhibition and no inhibition. (b) Comparison of the adsorption capacity of the peptide epitopes

22

368

23

after 30 min of incubation at room temperature at 10 mM phosphate buffer at different pH values.

24

(c) Comparison of the strength of the interaction between

25

RRPFNIGINNQQ379 by NP12. In addition to the control (left), two scenarios are illustrated, RRPFNIGINNQQ379 (80 µg/mL) and 865NITIHITDTNNK876 (80 µg/mL) on NP12 (0.5 mg/mL)

mg/mL) and

26

values

by

865

NITIHITDTNNK

UV-vis

RRPFNIGINNQQ379 (0.005-0.22

(0.2 mg/mL) in 10 mM phosphate buffer at different pH

spectroscopy.

(d)

Determination

the

binding

strength

of

27

Comparison of the amount of

29

presence and absence of NP12. The experimental conditions were as follows: NP12, 0.5 mg/mL;

30

concentration ratio of

31

80/120, 80/160, 120/120 and 160/160, respectively; pH 8.0 10 mM phosphate buffer.

368

RRPFNIGINNQQ 865

379

of

28

to

368

368

865

NITIHITDTNNK

876

876

in pH 8.0 10 mM phosphate buffer by HPLC. (e)

NITIHITDTNNK876 bound to

RRPFNIGINNQQ379 in the

RRPFNIGINNQQ379 (µg/mL) to 865NITIHITDTNNK876 (µg/mL), 80/80,

19

368



Page 20 of 41

1

368

RRPFNIGINNQQ379 by NP12 was calculated as follows:

M1 − M 2 × 100 M1

R(%) =

2

3

Where R represents the ratio of the binding sites inhibited by NP12, M1 and M2

4

represents the amount of

5

(wt/wt ratio) in the control and experimental group, respectively.

865


368

RRPFNIGINNQQ379

The analysis of the competitive inhibition experiment would be valid if the

6 7

following assumptions were met: (1) strong binding of

8

NP12; (2) no or very weak binding of

9

interaction between

368

865

368

RRPFNIGINNQQ379 to

NITIHITDTNNK876 to NP12; (3) strong

RRPFNIGINNQQ379 and

865

NITIHITDTNNK876. Additional

10

experiments were carried out to establish conditions where these three prerequisites

11

are

12

368

13

It was noted that the amount of 865NITIHITDTNNK876 adsorbed by NP12 was similar

14

to that of

15

experiment cannot be carried out at this pH value. However, the uptake of

16

865

17

8.0 to 12.0, although the adsorption capacity for

18

optimum over this pH range. These conditions are very close to the alkaline midgut

19

environment of the insect larvae.72 Therefore, the prerequisites (1) and (2) for the

20

competitive inhibition experiment can be fulfilled at pH 8.0-12.0. The interaction

21

between

22

UV-vis spectroscopy. A series of mixed samples containing the two peptide epitopes

satisfied

(Figure

5b-d).

Figure

5b

compares

amount

of

RRPFNIGINNQQ379 and 865NITIHITDTNNK876 absorbed on NP12 at different pH.

368

RRPFNIGINNQQ379 at pH 6.0. Therefore, the competitive inhibition

NITIHITDTNNK876 was much lower than that of the 368RRPFNIGINNQQ379 at pH

865

NITIHITDTNNK876 and

368

368

RRPFNIGINNQQ379 was not

RRPFNIGINNQQ379 was investigated by

20

the


Page 21 of 41 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

were scanned between 190-400 nm after 1 h of incubation at 25 °C. As controls,

2

single samples of these two peptides were also treated in a similar procedure. The UV

3

absorbance spectra of the peptide-peptide complexes and their respective pure

4

controls are shown in Figures S7-9. Figure 5c compares the intermolecular force

5

between

6

368

7

was estimated as follows:

these

two

peptides

at

different

pH

and

different

RRPFNIGINNQQ379/865NITIHITDTNNK876 ratios. The strength of the interaction

ΔA = A368 RRPFNIGINNQQ379 + A865 NITIHITDTNNK876 − A368 RRPFNIGINNQQ379 +865 NITIHITDTNNK876

8

9

Where A368 RRPFNIGINNQQ379 and A865 NITIHITDTNNK876 represent the absorbance values of the

10

pure controls of these two peptides at their respective maximum adsorption

11

wavelength, and A368 RRPFNIGINNQQ379 +865 NITIHITDTNNK876 is the absorbance value of the

12

mixed samples of these two peptides. The intermolecular interaction can be

13

determined by this method is that the formation of peptide-peptide complexes will

14

lead to a decrease in absorbance values. As shown in Figure 5c, the interaction

15

between

16

368

17

from 6.0 to 12.5. The

18

8.0 was then quantified by HPLC (Figure 5d). A series of mixed samples of these

19

two peptides were centrifugal filtrated (omega membrane MWCO 100 KDa) after 1 h

20

of incubation at this pH in 10 mM phosphate buffer. The residual concentration of

21

these two peptides in the filtrate was detected by HPLC. As controls, single samples

22

of both peptides were also operated with an identical procedure. The affinity between

these

two

peptides

increased

with

ratio

of

RRPFNIGINNQQ379/865NITIHITDTNNK876, and decreased with increasing pH 865

NITIHITDTNNK876-368RRPFNIGINNQQ379 affinity at pH

21

increasing



Page 22 of 41

1

these two peptides is obtained by comparing the residual 865NITIHITDTNNK876 in the

2

mixed samples with that in its pure controls. As shown in Figure 5d, the residual

3

level of

4

pure controls. This result indicates that the prerequisite (3): a strong specific binding

5

of 865NITIHITDTNNK876 to 368RRPFNIGINNQQ379 can be satisfied at pH 8.0.

865

NITIHITDTNNK876 in the mixed samples was much lower than that in its

The subsequent competitive inhibition experiment was performed at 10 mM pH

6 7

8.0 phosphate buffer solution. The concentration ratio of

8

865

9

amount of

368

RRPFNIGINNQQ379 to

NITIHITDTNNK876 was identical with that in Figure 5d. Figure 5e compares the 865


368

RRPFNIGINNQQ379 in the incubation

10

system with (experimental group) and without (control group) the addition of NP12. It

11

was found that the bound amount of

12

was significantly lower than that in the control group demonstrating that

13

preincubation of

14

865

15

significant,

16

368

17

160/500/160. The results indicate that NP12 and

18

the same domain(s) in

19

NP12 captures Bt Cry1Ab/Ac proteins via the epitope region

20

of these proteins, just as the cadherin-like protein Bt-R1 insect receptor (Figure 2)

21

establishing the biomimetic behavior of NP12 with the insect receptor cadherin-like

22

protein Bt-R1.

368

865

NITIHITDTNNK876 in the experimental group

RRPFNIGINNQQ379 with NP12 inhibited subsequent binding of

NITIHITDTNNK876 to varying

368

RRPFNIGINNQQ379. The inhibition by NP12 was

from

60%

to

84%

at

RRPFNIGINNQQ379/NP12/865NITIHITDTNNK876

368

865

a of

about

ratio

of

80/500/80

to

NITIHITDTNNK876 compete for

RRPFNIGINNQQ379. This supports the conclusion that

22

wt/wt/wt


368

RRPFNIGINNQQ379

Page 23 of 41 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

Specificity of NP12 to Bt Proteins. The selectivity of NP12 was

2

evaluated by comparing its affinity to Bt Cry1Ab and other Bt proteins, such as Bt

3

Cry1Ac, Cry2A, Cry1C and Cry1F proteins. These Bt Cry proteins have very similar

4

molecular weights (64-68 KDa) and highly similar isoelectric points (≈6, program

5

Theoretical pI/MW, ExPASy Proteomics tools). The selective adsorption experiment

6

was performed in 10 mM pH 8.0 phosphate buffer, conditions where all proteins have

7

good solubility. As shown in Figure 6, the affinities of NP12 to these Bt Cry proteins

8

were very different although their molecular weights, isoelectric points and crystal

9

strucures are very similar. The results indicate that it is not the overall charge of the

10

whole protein but rather some specific peptide residues that are complementary to

11

domains in NP12 that are responsible for protein-NP binding.[16,18] NP12 exhibited the

12

highest adsorption capacity for the Bt Cry1Ab protein. This can be attributed to the

13

screening process that was used to select NP12, affinity to 368RRPFNIGINNQQ379, an

14

epitope in domain II loop 2 of the Bt Cry1Ab protein. NP12 also displayed high

15

affinity and selectivity for the Bt Cry1Ac protein. This result is not surprising since Bt

16

Cry1Ac and Bt Cry1Ab proteins share very high degrees of sequence and structural

17

homology. For example, there is almost 100% sequence identity in domain I, 98%

18

sequence identity in domain II and 45% sequence identity in domain III for Bt

19

Cry1Ac and Cry1Ab proteins.[73] Moreover, the amino acid sequence in the most

20

important receptor binding region (domain II loop 2) includes the epitope

21

368

22

NP12 showed very weak affinity to Bt Cry2A, Cry1C and Cry1F

RRPFNIGINNQQ379, is identical for Bt Cry1Ac and Cry1Ab proteins. Finally,

23



5 0 ng /m L

50

40

30

B in d in g c a p a c ity ( µg /g )

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

Page 24 of 41

20

10

0 B t C ry1A b

B t C ry1A c

B t C ry2A

B t C ry1C

B t C ry1F

S elec tiv ity 1 2

Figure 6． Comparison of the selectivity of NP12 to Bt Cry1Ab, Cry1Ac, Cry2A, Cry1C and

3

Cry1F proteins. The experimental conditions were as follows: Bt proteins, 50 ng/mL; NP12, 0.5

4

mg/mL; incubation at pH 8.0 10 mM phosphate buffer for 2 h at room temperature.

5 6

proteins. These proteins show very low sequence and structural homologies with the

7

Bt Cry1Ab protein although their isoelectric points are very similar. For example, Bt

8

Cry2Aa protein has only 17% amino acid sequence identity with Bt Cry1Ab protein.

9

Moreover, the receptor binding epitopes for Lepidopteran and Dipteran, as suggested

10

from the crystal structure of Bt Cry2Aa, were comprised of a distribution of

11

hydrophobic residues (Ile474–Ala477 from β12a, Val365–Leu369 from the β5-β6

12

loop, and Leu402–Leu404 from the β7-β8 loop) across the solvent-exposed surface of

13

the β prism and β sandwich domains. These receptor binding epitopes were very

14

different from that of other Bt Cry proteins.74 The receptor binding properties of Bt

24


Page 25 of 41 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

Cry2Aa protein was also different from that of Bt Cry1Ab and Cry1Ac proteins. For

2

example, Bt Cry1Ab and Cry1Ac proteins recognize the same binding site in

3

Helicoverpa zea and Pectinophora gossypiella midgut brush border membrane

4

vesicles, while Bt Cry2Aa does not.75 Similarly, the Bt Cry2A family (Cry2Aa,

5

Cry2Ab, and Cry2Ae) compete for a common binding site in the midgut of

6

Helicoverpa species, but which was not shared by the Bt Cry1Ac protein.76 The

7

difference in sequence and structural similarity presents an entirely different binding

8

interface with reduced affinity of Bt Cry2A protein to the abiotic receptor NP12. This

9

explanation might be extended to Bt Cry1C and Cry1F proteins although the crystal

10

structure of these two Bt proteins have not been reported, and the receptor binding

11

mechanisms are not well understood. However, it is not unreasonable to assume that

12

once the epitopes of these Bt proteins are identified, it will be possible to develop

13

abiotic affinity ligands for this group using the biomimetic strategy described in this

14

work.

15

Adsorption Kinetics and Isothermal Analysis. Figure 7a shows the

16

kinetic adsorption curve of Bt Cry1Ab protein on NP12. The kinetic adsorption data

17

were analyzed using the Lagergren pseudo-first-order and pseudo-second-order

18

kinetic models in order to examine the controlling mechanism of the adsorption

19

processes between Bt Cry1Ab and NP12. The fitting parameters were shown in Table

20

S1. The kinetic adsorption experiments showed that the time required for reaching

21

adsorption equilibrium was approximately 40 min for Bt Cry1Ab at 10 mM phosphate

22

buffer (pH 6.0). The rate constant for the pseudo-first-order and pseudo-second-order

25



70 60

B t C ry1A b

50

p s eu d o -‐firs t-‐o rd er fit p s eu d o -‐s ec o n d -‐o rd er fit

40

A d s o rp tio n a m o u n ts ( µg /g )

80

30 20 10

(a)

0 0

20

40

60

80

100

120

t (m in ) 1 100

80

B t C ry1A b

L an g m u ir fit F reu d lic h fit

60

A d s o rp tio n c a p a c ity ( µg /g )

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

Page 26 of 41

40

20

(b )

0 0

10

20

30

40

50

60

C o n c en tratio n ( µg /L ) 2 3

Figure 7. The kinetic (a) and isothermal (b) adsorption curves of Bt Cry1Ab protein on NP12. The

4

conditions for the kinetic adsorption experiments were as follows: Bt Cry1Ab proteins, 50 ng/mL;

5

NP12, 0.5 mg/mL; incubation at pH 6.0 10 mM phosphate buffer solution for 5-120 min at room

6

temperature. The conditions for the isothermal adsorption experiments were as follows: Bt

7

Cry1Ab proteins, 0-60 ng/mL; NP12, 0.5 mg/mL; incubation at pH 6.0 10 mM phosphate buffer

8

solution for 2 h at room temperature.

9 26


Page 27 of 41 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

kinetic model was 0.42 min -1 and 0.017 g µg -1 min -1 , respectively. The

2

pseudo-second-order model (r2=0.9969) was more suitable to describe the adsorption

3

kinetic data than the pseudo-first-order model (r2=0.9900). Figure 7b represents the

4

isothermal adsorption curve of Bt Cry1Ab protein on NP12. The equilibrium

5

adsorption data were fitted using the Langmuir and Freundlich adsorption isothermal

6

models. As shown in Table S1, the equilibrium adsorption data fitted better to the

7

Langmuir adsorption isothermal equation (r2=0.9856) than the Freundlich adsorption

8

isothermal model (r2=0.8052). The maximum theoretical adsorption capacity

9

calculated from the fitting results was about 91 µg g-1. The equilibrium dissociation

10

constant (Kd) was about 1.7 µg L-1 (0.026 nM), which was close to that obtained

11

between Bt Cry1Ab protein and natural or recombinant Bt-R1 (~ 1 nM).77,78 These

12

results highlight the similarities of behavior of NP12 and the wild type receptor.

13

pH-Responsive Catch and Release of Bt Cry1Ab Proteins. NIPAm

14

copolymers containing carboxylate groups are known to be pH responsive.79,80 The

15

feasibility of binding and releasing Bt Cry1Ab protein by exploiting the

16

pH-responsive property of NP12 was investigated in detail. Figure 8a shows the

17

procedure for evaluating the binding (pH 6.0) and release (pH 10.0) of Bt Cry1Ab

18

proteins. The residual Bt Cry1Ab proteins following adsorption and desorption as a

19

function of pH are shown in Figure 8b. Bt Cry1Ab proteins were captured by NP12 at

20

pH 6.0, and released from NP12 at pH 10.0 (Figure 8b). Moreover, although the

21

binding percent was just 50% for Bt Cry1Ab protein under these conditions, the

22

amount of protein captured can be increased by simply adding more

27



1

2 3 4

B t C ry1A b

12

10

8

R e s id u a l c o n c e n tra tio n ( µg /L )

14

6

4

2

0

5

In itial (p H 6.0)

A fter ad s o rp tio n (p H 6.0)

A fter d es o rp tio n (p H 10.0)

(b )

6

1200

7 8 9 10

N P 12

800

H y d ro d y n a m ic d ia m e te r (n m )

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

Page 28 of 41

400

0 4

11

6

pH

8

10

(c )

12

Figure 8. (a) Schematic of the pH-responsive catch and release of Bt Cry1Ab proteins (12 µg/L)

13

by NP12 (0.5 mg/mL). The catch and release experiment was performed at pH 6.0 and 10.0

14

phosphate buffer solution (10 mM), respectively. (b) Comparison of the residual Bt Cry1Ab

15

proteins after the adsorption and desorption processes. (c) The hydrodynamic diameters of NP12

16

at 25°C as a function of the pH of the phosphate buffer (10 mM). (d) Illustration of the

17

pH-responsive “catch-and-release” of Bt Cry1Ab proteins by NP12 by switching the pH from 6.0

18

to 10.0.

19 20

NPs. We suggest that there are several factors that contribute to this pH responsive

21

behavior. Electrostatic interaction between the arginine residues on Bt Cry1Ab and the

22

AAc groups on NP12 are important for protein-NP binding. An increase in pH from

23

pH 6.0 to 10.0, results in a decrease in this electrostatic interaction. This is due in part

24

to a decrease in the fraction of protonated guanidinium groups in the arginine residues

25

(pKa 12.5) compared to those at pH 6 resulting in a decrease in adsorption capacity of 28


Page 29 of 41 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

Bt Cry1Ab proteins on NP12 at higher pH value (Figure S5). A change in pH also

2

influences the NP; the nanoparticle size increases significantly from pH 4.0 to 8.0

3

(Figure 8c) which corresponds to the range of pKa’s of the carboxylic acid groups in

4

the NP. The volume reaches a plateau at pH above 8.0. The volume increase can be

5

attributed to changes in the Donnan potential and osmotic pressure inside the

6

nanoparticles.81

7

“catch-and-release” of Bt Cry1Ab proteins by NP12 as the pH changes from 6.0-10.0

8

(Figure 8d). At pH 6.0, NP12 exists in a more collapsed, less solvated state. At pH

9

10.0, NP12 now exists in a solvent swollen state.82 As a consequence, the charge

10

density per unit volume inside the nanoparticles decreases. These changes, in

11

conjunction with the reduced positive charge of Bt Cry1Ab binding domain weaken

12

the NP12-Bt Cry1Ab interaction, triggering the release of Bt Cry1Ab protein from

13

NP12.

The impact of these changes on protein binding is a

14

Separation of Bt Cry1Ac Protein from Transgenic Bt Cotton

15

Leaves. The applicability of NP12 for concentrating Bt Cry1Ac protein from the

16

extract of transgenic Bt cotton leaves was evaluated. The extraction and separation

17

procedure is outlined in Figure 9. The concentration of Bt Cry1Ac protein and total

18

protein in the leaf extract, the flow-through and the eluant fractions of the treated and

19

fresh transgenic Bt cotton leaves are given in Table 2. The amounts of Bt Cry1Ac

20

proteins and total proteins in transgenic Bt cotton leaves were approximately 36-42

21

ng/g and 26-41 mg/g, respectively. As can be seen from Table 2, NP12 was capable

22

of sequestering and releasing Bt Cry1Ac protein effectively from the cotton leaf

29



Page 30 of 41

1

2 3

Figure 9. Schematic of the extraction of Bt Cry1Ac proteins from transgenic Bt cotton leaves, and

4

the separation of Bt Cry1Ac proteins from the cotton leaf extract by NP12 (0.5 mg/mL). The

5

extraction of Bt Cry1Ac from transgenic Bt cotton leaves was performed at 10 mM phosphate

6

buffer with 0.5% tween-20 (pH 6.0). The capture of Bt Cry1Ac from the cotton leaf extract by

7

NP12 was performed at 10 mM phosphate buffer (pH 6.0). The release of the bound Bt Cry1Ac

8

from NP12 was performed at 10 mM phosphate buffer (pH 10.0).

9 10

Table 2 The concentration of the Bt Cry1Ac protein and total protein in the extract, the

11

flow-through fraction and the eluant fraction of the treated and fresh transgenic Bt cotton leaves Treated leaves Bt Cry1Ac Amount in transgenic Bt cotton leaves (mg/g) Concentration in leaf extract (C1, mg/mL) Concentration in flow-through fraction (C2, mg/mL) Concentration in eluant fraction (C3, mg/mL) Binding ratio (%) Elution ratio (%) Recovery (%)

4.20×10

-5

1.05×10

-5

2.15×10

-6

2.68×10

-6

a

b

c

Total protein

Bt Cry1Ac

Total protein

3.58×10

-5

26.3

9.00×10

-6

6.57

2.15×10

-6

1.03

0.34

2.15×10

-6

0.21

58.9

79.8

52.0

68.8

86.7

8.32

92.4

9.43

51.1

6.64

48.1

6.48

12

a

1 1 Binding ratio (%) = ( × C1 × 0.5 − C2 × 0.5) /( × C1 × 0.5) ×100 2 2

13

b

1 Elution ratio (%) = (C3 × 0.5) /( × C1 × 0.5 − C2 × 0.5) ×100 2

14

c

1 Recovery (%) = (C3 × 0.5) /( × C1 × 0.5) ×100 2

41.1 10.3 1.04

15

30

Fresh leaves


Page 31 of 41 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

extract by capturing at pH 6 and releasing at pH 10.0. Although the binding ratio of

2

the interfering proteins to NP12 at pH 6.0 was as high as that of the Bt Cry1Ac protein,

3

the elution ratio of the bound interfering proteins at pH 10.0 was much lower than that

4

of the Bt Cry1Ac protein. Therefore, the relative content of Bt Cry1Ac protein in the

5

extracted total proteins was increased by an extra cycle of catch and release step

6

although the initial concentration of the interfering proteins was significantly larger

7

than the Bt Cry1Ac protein. The purity of Bt Cry1Ac protein in the elution fraction

8

was approximately 7.5 times higher than that in the leaf extract. The purity could be

9

higher if more cycles of catch and release were performed. The recovery of the Bt

10

Cry1Ac protein and the interfering proteins from the leaf extract was estimated to be

11

50% and 6.5%, respectively. Therefore, NP12 could be used as an abiotic affinity

12

ligand in separation and concentration of Bt Cry1Ab/Ac protein from transgenic Bt

13

crops.

14 15

n CONCLUSIONS

16

In conclusion, we demonstrate a novel strategy for the discovery of a synthetic

17

polymer nanoparticle that can selectively bind and capture Bt Cry1Ab/Ac proteins

18

from complex biological matrices. The strategy is based on identifying a synthetic

19

polymer NP with engineered affinity and selectivity to a specific peptide epitope of

20

the Bt Cry1Ab/Ac protein. This same epitope is involved in the binding of Bt

21

Cry1Ab/Ac proteins to the insect receptor cadherin-like protein Bt-R1. Thus the NP

22

selected by this strategy mimics the insect receptor cadherin-like protein Bt-R1. In this

31



1

work, a small library of polymer hydrogel NPs containing different charged,

2

hydrophilic and hydrophobic groups were synthesized and screened for binding to the

3

three epitopes of Bt Cry1Ab/Ac,

4

368

5

loop 3). NP12, a 2% BIS cross-linked hydrogel containing 40% TBAm, 18% AAm

6

and 40% AAc was found to have high affinity to the amino acid sequence

7

368

8

proteins. This NP also bound both Bt Cry1Ab and Bt Cry1Ac, proteins that

9

incorporate the epitope 368RRPFNIGINNQQ379 in their molecular structure. In contrast,

10

the affinity of NP12 was very low for Bt Cry2A, Bt Cry1C and Bt Cry1F proteins.

11

These three closely related proteins contain epitopes that are involved in Bt

12

protein-insect receptor binding, however these epitopes have very low sequence

13

identity from those of the Bt Cry1Ab/Ac proteins. This result validates our initial goal

14

of engineering the synthetic polymer NPs as Bt Cry1Ab/Ac protein affinity ligands

15

using a biomimetic strategy. To help identify the location of the dominant NP-Bt

16

Cry1Ab/Ac protein interaction, a competitive inhibition experiment was performed

17

using NP12 as an inhibitor to block the binding of 865NITIHITDTNNK876 (recognition

18

site in insect receptor cadherin-like protein Bt-R1) to

19

shown that the interaction between these two peptides was significantly inhibited by

20

the addition of NP12 to the incubation solution. This result supports the notion that a

21

similar molecular recognition mechanism for NP12 and the cadherin-like protein

22

insect receptor in binding to Bt proteins is operating and that the amino acid sequence

280

FRGSAQGIEGS290 (domain II loop α8),

RRPFNIGINNQQ379 (domain II loop 2) and

436

FRSGFSNSSVSIIR449 (domain II

RRPFNIGINNQQ379 representing the epitope in domain II loop 2 of Bt Cry1Ab/Ac

368

32


RRPFNIGINNQQ379. It was

Page 32 of 41

Page 33 of 41 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

368

RRPFNIGINNQQ379 located in domain II loop 2 is the dominant binding site of Bt

2

Cry1Ab/Ac proteins on NP12. It was also found that the Bt Cry1Ab/Ac protein

3

affinity could be modulated by pH. This behavior allows for the capture and release of

4

Bt Cry1Ab/Ac proteins by adjusting the pH from 6.0 to 10.0. This increases the utility

5

of using NP12 for recognition, capture and concentration of Bt Cry1Ab/Ac proteins

6

from both the biological and environmental medium, such as the culture of Bt or

7

recombinant Bt strains, the plant tissues and the rhizosphere soil of the transgenic Bt

8

crops.

9 10

n ASSOCIATED CONTENT

11

S Supporting Information ○

12

The Supporting Information is available free of charge on the ACS Publications

13

website.

14 15

n AUTHOR INFORMATION

16

Corresponding Author

17

[email protected]; [email protected]

18

Notes

19

The authors declare no competing financial interest.

20 21

n ACKNOWLEDGEMENTS

22

This work was financially supported by the National Natural Science Foundation of

33



Page 34 of 41

1

China (Grants No. 21577044 and 21177047), the Program for New Century Excellent

2

Talents in University (Grant No. NCET-13-0808), the Fundamental Research Funds

3

for the Central Universities (Programs No. 2014PY019 and 2013PY138), the Wuhan

4

Youth Science and Technology Chenguang Program (Grant No. 201271031378), the

5

Natural Science Foundation of Hubei Province of China (Grant No. 2014CFA016),

6

the

7

2016ZX08001001), and the US National Science Foundation (Grant No.

8

DMR-1308363).

National

Special

Key

Project

for

Transgenic

Breeding

(Grant

No.

9 10

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Biomimicry strategy

Bt protein―insect receptor protein binding

Bt protein―polymer NP binding


A synthetic polymer affinity ligand for Bacillus thuringiensis (Bt

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