Molecular Sieving on the Surface of a Nano-Armored Protein

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Molecular Sieving on the Surface of a Nano-Armored Protein Bibifatima Kaupbayeva, Hironobu Murata, Amber Lucas, Krzysztof Matyjaszewski, Jonathan S. Minden, and Alan J Russell Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01651 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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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

Molecular Sieving on the Surface of a NanoArmored Protein Bibifatima Kaupbayeva1,2, Hironobu Murata2, Amber Lucas1,2, Krzysztof Matyjaszewski2,3, Jonathan S. Minden1,2,4, Alan J. Russell a1,2,3,4,5,6* 1Department

2Center

of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, United States

for Polymer-Based Protein Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213, United States

3Department

of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, United States

4Department

5Department

6Disruptive

of Biomedical Engineering, Scott Hall 4N201, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213, United States

of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213, United States

Health Technology Institute, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213, United States

KEYWORDS avidin, biotin, ATRP, molecular sieving

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ABSTRACT

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The molecular sieving properties of protein surface-attached polymers are the central features

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in how polymers extend therapeutic protein lifetimes in vivo. Yet, even after thirty years of

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research, permeation rates of molecules through polymer-surrounded protein surfaces are

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largely unknown. As a result, the generation of protein-polymer conjugates remains a

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stochastic process, unfacilitated by knowledge of structure-function-polymer architecture

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relationships. In this work, polymers are grown from the surface of avidin using atom transfer

54

radical polymerization (ATRP) and used to determine how polymer length and density

55

influence the binding kinetics of ligands as a function of ligand size and shape. The rate of

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binding is strongly dependent on the grafting density of polymers and the size of the ligand,

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but interestingly, far less dependent on the length of the polymer. This study unveils a deeper

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understanding of relationship between polymer characteristics and binding kinetics,

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discovering important steps in rational design of protein-polymer-conjugates.

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INTRODUCTION

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Protein-polymer conjugates are unique macromolecules that combine the rugged

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attractiveness of synthetic chemistry and the exquisite balance of activity and specificity

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found in biological systems. Since the synthesis of the first protein-polymer conjugate was

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reported in 1977, the application of protein-polymer conjugates has expanded significantly1.

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Today these conjugates are used in biotechnology2, cosmetics3, foods, surface coatings and

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therapeutics4. Protein-polymer conjugates can be synthesized using two different strategies:

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“grafting to” or “grafting from”. The process of “grafting to” consists of covalent attachment

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of pre-synthesized and characterized polymers to the protein5. A limitation of this method has

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been low achievable grafting densities of polymers on protein surfaces due to steric hindrance

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created by subsequently attached polymer chains. Also, control of the attachment site location

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and purification of the resulting conjugates can be challenging6-8. In the “grafting from”

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approach, the polymers are generated from the protein surface by controlled radical

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polymerization (CRP). Most commonly, either atom transfer radical polymerization (ATRP)

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9-11

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The “grafting from” method, has enabled tighter control over modification site, high grafting

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density and simplified purification. Since the number and molecular weight of polymer chains

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are predetermined, the method allows the generation of protein-polymer conjugates with low

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dispersity (D).

or reversible addition-fragmentation polymerization (RAFT)12-15 methods have been used.

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Polymer-based protein engineering has been used prepare conjugates with enhanced

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pH and temperature stability, tailored substrate affinity and stability in organic environments8,

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11, 16-20.

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conjugates” by anchoring stimuli responsive polymers that respond to temperature and pH21-24.

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However, our understanding of how the polymer layer affects substrate diffusion limits and

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rates to the active site of proteins is limited. Polymers sieving properties are important criteria

In recent years, considerable attention has been paid to the creation of “smart

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affecting the efficacy of protein drugs25 and biomedical devices26. For example, enzyme-

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polymer conjugates used in therapy need to repel protein-antibody interactions and protease-

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mediated hydrolysis, while allowing proteins to interact with their substrates and their ligands.

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In 2012 Gauthier and colleagues, in an elegant study, demonstrated that comb-shaped

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poly(oligo(ethylene glycol) methacrylate) pOEGMA polymers can create a molecular sieving

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effect when grafted from a chymotrypsin surface by blocking larger macromolecules27. But,

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to the best of our knowledge, no one has been able to determine the rate at which molecules

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penetrate the polymer shell grown around proteins.

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The term ‘molecular sieving’, as proposed by Gauthier27, suggests a threshold for

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‘sieving size’, below which diffusion of molecules is unhindered, and above which it becomes

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hindered. In this study, we use the term ‘molecular sieving’ to describe polymer-mediated

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shielding of binding sites that impact the permeation rates of ligands to the protein surface.

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Protein-ligand interactions have often been studied with avidin-biotin complexes28-30.

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Avidin is a tetrameric protein purified from egg white that binds biotin with exquisite strength

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and speed. The high affinity of avidin towards biotin allowed us to biotinylate molecules of

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different shapes and sizes and monitor their permeation rates through covalently-attached

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polymer layers of varying lengths and densities. By using ATRP to decorate avidin with

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poly(carboxybetaine methacrylate) (pCBMA) polymers, a zwitterionic polymer that has non-

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fouling properties, we have created a well-controlled system for understanding the

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relationship between polymer length/density and the diffusion/accessibility of different

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size/shape ligands31-35.

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The number of chains grown from a protein using “grafting from” ATRP with amino-

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reactive, single-headed initiators cannot exceed the number of accessible amine groups on the

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surface of the protein. To overcome this limitation and better understand the impact of

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polymer density on the rate of sieving by attached polymers, we designed a novel, NHS4 ACS Paragon Plus Environment

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functionalized, double-headed ATRP initiator that supported the growth of two polymers

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from one initiation point. Eight different molecular weight avidin-pCBMA conjugates were

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synthesized to study the penetration rate of molecules through the polymer shell to the protein

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binding site as a function of polymer chain length, polymer grafting density, ligand size, and

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ligand shape. Polymers grafting density and ligand size have a profound effect on the rate of

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binding of ligands to a protein shielded with covalently attached polymers. Surprisingly, the

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molecular weight of the polymer attached to the protein and shape of the diffusing molecule

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have a small impact on the rate of ligand binding.

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EXPERIMENTAL SECTION

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Materials. Avidin from egg white was purchased from Lee Biosolutions (Maryland

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Heights, MO). Aprotinin and Histone were purchased from Sigma Aldrich (St. Louis, MO).

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Horse Radish Peroxidase was purchased from Millipore Sigma (Burlington, MA). Biotin-

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PEG-NHS was purchased from Thermo Fisher (Waltham, MA). Biotin-PEG was purchased

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from Creative PEG Workers (Chapel Hill, NC). Single ATRP initiator was synthesized as

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described earlier18. Initiation inhibitor was prepared as described previously36.

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NHS-Functionalized double-headed ATRP initiator synthesis. Double-headed

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ATRP initiator was synthesized as follows. N,N’-dicyclohexylcarbodimine (10.9 g, 53 mmol)

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in dichloromethane (10 mL) was slowly added to the solution of 2-bromo-isobutyric acid (8.0

152

g, 48 mmol) and N-hydroxysuccinimide (6.1 g, 53 mmol) in dichloromethane (100 mL) at

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0 °C. Mixture was stirred at room temperature overnight. Precipitated urea was filtered out

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and the filtrate was evaporated to remove solvent. 2-bromo-2-methylpropionyl-N-

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oxysuccinimine ester was isolated by recrystallization in 2-propanol. Next, 2-bromo-2-

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methylpropionyl-N-succinimide ester (5.3 g, 2.0 mmol) was slowly added to the solution of

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diethylenetriamine (1.0 g, 9.7 mmol) and triethylamine (1.4 mL, 1.0 mmol) in acetonitrile (50

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mL) at 0 °C. The mixture was stirred at room temperature overnight. 5 ACS Paragon Plus Environment

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hydroxysuccinimide was filtered out and the filtrate was evaporated to remove solvent. Ethyl

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acetate (50 mL) was added to the mixture and the organic phase was washed with 50 wt%

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sodium carbonate aq. (20 mL × 3) and saturated NaCl aq. (20 mL × 3). The organic phase was

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dried

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methylpropanamido)ethylamine was isolated by column chromatography (silica and

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acetonitrile). Succinic anhydride (600 mg, 6.0 mmol) and triethylamine (840 µL, 6.0 mmol)

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was added to the solution of bis(2-(2-bromo-2-methylpropanamido)ethylamine (2.2 g, 5.5

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mmol) in acetonitrile (50 mL), then mixture was stirred at room temperature overnight. After

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solvent was evaporated, ethyl acetate (50 mL) was added to the mixture. The organic phase

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was washed with 1 N HCl aq. (20 mL × 3) and saturated NaCl aq. (20 mL × 3). The organic

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phase was dried with MgSO4 and evaporated to remove solvent. To the solution of bis(2-(2-

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bromo-2-methylpropanamido)ethyl)amino)-4-oxobutanoic acid (2.0 g, 2.0 mmol) in

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acetonitrile (50 mL), di(N-succinimidyl) carbonate (1.1 g, 4.3 mmol) and triethylamine (560

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µL, 4.0 mmol) were added and the mixture was stirred at room temperature overnight. After

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the solvent evaporated, double-headed ATRP initiator was isolated by column

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chromatography (silica, acetone:chloroform (1/4 volume ratio)) The chemical structures were

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confirmed by 1H and 13C NMR and IR. See Supplementary Methods.

with

Na2CO3

and

evaporated

to

remove

solvent.

Bis(2-(2-bromo-2-

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Attachment of single ATRP initiator on the surface of avidin. Synthesis of the

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ATRP initiator was carried out as described previously18. After synthesis, the initiator (523

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mg, 1.56 mmol) and avidin (500 mg, 0.03 mmol protein, 0.31 mmol primary amine groups)

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were dissolved in 0.1 M sodium phosphate buffer, (pH 8, 100 mL). The solution was stirred at

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4 °C for 2 h and avidin conjugates were purified by dialysis using 15 kDa molecular mass

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cutoff dialysis tube, in 25 mM sodium phosphate (pH 8), for 24 h at 4°C and then lyophilized.

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Double-headed ATRP initiator attachment onto avidin surface: Following the synthesis,

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double-headed ATRP initiator (935 mg, 1.56 mmol) was dissolved in DMSO (4 mL) added to 6 ACS Paragon Plus Environment

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a solution of avidin (500 mg, 0.31 mmol primary amine groups) in 0.1 M sodium phosphate

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buffer (pH 8, 100 mL). The mixture was stirred at 4°C and for 2 h, then dialyzed against 25

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mM sodium phosphate buffer (pH 8), using dialysis tubing with molecular mass cutoff of 15

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kDa, for 24 h at 4°C and then lyophilized.

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MALDI-ToF analysis. MALDI-ToF measurements were recorded using a PerSeptive

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Voyager STR MS with nitrogen laser (337 nm) and 20 kV accelerating voltage with a grid

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voltage of 90 %. 500 laser shots covering the complete spot were accumulated for each

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spectrum. For determination of molecular weights of synthesized protein-initiator complexes,

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sinapinic acid (10 mg mL-1) in 50% acetonitrile with 0.4% trifluoroacetic acid was used as

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matrix. Protein solution (1.0 mg mL-1) was mixed with an equal volume of matrix and the

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resulting mixture (2 µL) was loaded onto a silver sterling plate. Apomyoglobin, cytochrome C,

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and aldolase were used as standard calibration samples. ATRP initiator modification was

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determined by subtracting the native protein m/z values from protein-initiator conjugates m/z

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and dividing by the molecular weight of the initiator (220.5 g mol-1 for single and 478 g mol-1

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for double-headed initiators).

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SDS-PAGE analysis. 10µL of Avidin, Avidin-Br, or Avidin-Br2 solution (1.5mg/ml

200

in PBS) were mixed with 10µL of 2X Laemmli loading buffer. 10µL of samples were then

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loaded into wells on a 4%-15% precast gel and run for 35min at 200V. The gel was then

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washed and stained with PageBlue staining solution , and de-stained overnight.

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Trypsin digestion of avidin-initiator conjugates. Trypsin digests were used to

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generate peptide fragments from which initiator attachment sites could be determined using

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electrospray ionization mass spectrometry. Samples were digested according to the protocol

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described in the In-Solution Tryptic Digestion and Guanidination Kit. Protein or protein-

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initiator complexes (20 µg) (10 µL of a 2 mg mL-1 protein solution in deionized water) were

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added to of 50 mM ammonium bicarbonate (15 µL) with 100 mM dithiothreitol (1.5 µL) in a 7 ACS Paragon Plus Environment


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Eppendorf tube. The reaction was incubated for 5 min at 95 °C. Thiol alkylation was achieved

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by the addition of 100 mM iodoacetamide aqueous solution (3 µL) to the protein solution

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following by 20 min incubation in the dark for 20 min at room temperature. Following the

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incubation, trypsin (1 µL of 100 ng) was added to the protein solution and the reaction was

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incubated at 37 °C for 3 h. Then, an additional trypsin (1 µL of 100 ng) was subsequently

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added. The reaction was terminated after 2 h by the addition of trifluoroacetic acid (TFA).

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Digested samples were purified using ZipTipC18 microtips and eluted with matrix solution

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(200 µL of 50% acetonitrile with 0.1% formic) for subsequent ESI-MS analysis. The

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molecular weight of the expected peptide fragments before and after digestion was predicted

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using PeptideCutter (ExPASy Bioinformatics Portal, Swiss Institute of Bioinformatics).

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ESI-MS analysis. ESI-MS measurements were taken by using a Finnigan LCQ 37

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(Thermo-Fisher) quadrupole field ion trap mass spectrometer with electrospray ionization

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source. Each scan was acquired over the range m/z 150-2000 by using a step of 0.5 u, a dwell

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time of 1.5 ms, a mass defect of 50 pu, and an 80-V orifice potential. Samples at a protein

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concentration of 50 µM and eluted using 50% acetonitrile and 0.1% formic acid at a flow rate

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of 20 µL min-1.

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ATRP from single-headed ATRP initiator modified avidin. To synthesize avidin-

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pCBMA conjugates the final stoichiometry of 1:10:1:12 Initiator:Cu:NaAsc:Ligand was used.

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Avidin-initiator complex (50 mg, 0.0226 mmol of initiator groups) and CBMA monomer (259

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mg, 1.1 mmol for avidin-pCBMA50, 518 mg, 2.3 mmol for avidin-pCBMA100, 777 mg, 3.4

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mmol for avidin-pCBMA150 and 1036 mg, 4.5 mmol for avidin-pCBMA200) were dissolved in

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0.1 M sodium phosphate (45 mL). The flask was sealed with rubber septum and bubbled with

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nitrogen for 1 h.

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nitrogen for 20 min. Sodium ascorbate (300 µL of 20 mg mL-1, 0.03 mmol) and 1, 1, 4, 7, 10,

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10-hexamethyltriethylenetetramine (HMTETA) (100 µL, 0.37 mmol) were added to

In a separate flask, 50 mM CuCl2 solution (6 mL) was bubbled under


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deoxygenated CuCl2 solution and bubbled for another 5 min. Deoxygenated copper catalyst

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solution (5 mL) was added to a solution of deoxygenated avidin-Br/CBMA in 0.1 mM

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sodium phosphate (pH 8, 45 mL) and allowed to react for 1 h at room temperature. The

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reaction was stopped upon exposure to air and avidin-pCBMA conjugates were purified

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through dialysis (25 kDa MWCO) against 25 mM sodium phosphate for 24 hours at 4 °C and

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then lyophilized.

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ATRP from double-headed ATRP initiator modified avidin. For the synthesis of

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high-density conjugates the final stoichiometry of 1:10:1:12 Initiator:Cu:NaAsc:Ligand was

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used. Avidin-double-headed initiator conjugates (40 mg, 0.027 mmol initiator groups) and

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CBMA (310 mg, 1.35 mmol for avidin-pCBMA50, 619 mg, 2.7 mmol for avidin-pCBMA100,

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929 mg, 4.1 mmol for avidin-pCBMA150 and 1239 mg, 5.4 mmol for avidin-pCBMA200) were

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dissolved in sodium phosphate buffer (45 mL, 0.1 M, pH 8). The solutions of avidin-initiator

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conjugates and monomers were sealed with rubber septum and bubbled with nitrogen for 1 h.

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Sodium ascorbate (300 µL of 20 mg mL-1, 0.03 mmol) and HMTETA (100 µL) were added to

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deoxygenated CuCl2 (6 mL) of 50 mM and bubbled for 5 min. Deoxygenated copper catalyst

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(5 mL, 0.25 mmol Cu, 0.025 mmol NaAsc, 0.3 mmol HMTETA) was added to a solution of

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deoxygenated avidin-Br/CBMA and allowed to react for 1 hour at room temperature. The

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reaction was stopped upon exposure to air and avidin-pCBMA conjugates were purified

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through dialysis (25kDa MWCO) against 25 mM sodium phosphate for 24 hours at 4°C and

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then lyophilized.

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Cleavage of pCBMA from avidin surface. Avidin-pCBMA conjugates (20 mg) were

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placed in hydrolysis tubes and dissolved in 6 N HCl (6 mL). After five freeze-pump-thaw

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cycles, the hydrolysis was performed at 110 °C under vacuum for 24 hours. The cleaved

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polymers were dialyzed against deionized water at room temperature, using 1 kDa molecular



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mass cut off dialysis tubing and then lyophilized. The molecular weight and dispersity of

259

polymers were measured by gel permeation chromatography (GPC).

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BCA assay. Avidin conjugates were dialyzed against deionized water to remove salts

261

present in the samples and then lyophilized. Next, conjugates (1.0 mg) were dissolved in

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deionized water and the sample (25 µL) was mixed with bicinchonic acid (BCA) solution

263

(1.0) and copper (II) sulfate solution (50:1 vol:vol). The solution was incubated at 60°C for 15

264

min. Absorbance of the sample was recorded at 562 nm using UV-VIS spectrometer. Avidin

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concentration (wt%) was determined by comparison of the absorbance to a standard curve

266

(native avidin).

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Measuring conjugate hydrodynamic diameter. The DLS data was collected on a

268

Malvern Zetasizer nano-ZS. Native avidin and avidin conjugates (1.0 mg) were dissolved in

269

0.1 M sodium phosphate, (pH 8). The hydrodynamic diameter (Dh) of the samples was

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measured three times (12 runs/measurement). Reported values are number distribution

271

intensities.

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Protein biotinylation. For biotinylation, aprotinin (20 mg, 0.0031 mmol protein),

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histone (0.00093 mmol protein) and HRP (0.00045 mmol protein) were dissolved in 0.1 M

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sodium phosphate buffer (4 mL, pH 8). Biotin-PEG-NHS (18.2 mg, 0.031 mmol) for

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aprotinin, (5.4 mg, 0.0093 mmol) for histone and for HRP (2.6 mg, 0.0045 mmol) and were

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dissolved in DMSO (200 µL) and added to a protein solution. The solution was stirred at 4°C

277

and for 2 h and protein-Biotin conjugates were purified by dialysis using 15 kDa molecular

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mass cutoff dialysis tube in deionized water and then lyophilized.

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Fluorescamine assay. Fluorescamine assay was used to determine the biotinylation

280

extend of proteins. Protein-biotin samples (80 µL, 1.0 mg mL-1), 100 mM sodium phosphate

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(80 µL, pH 8.5), and fluorescamine solution in DMSO (40 µL, 3 mg mL-1) were added into a

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96-well plate and incubated at room temperature for 15 min. Fluorescence intensities were 10 ACS Paragon Plus Environment

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measured at the excitation of 390 nm and emission of 470 nm with 10-nm bandwidths by a H

284

Synergy plate reader. Biotinylation was determined by comparison of the fluorescence to the

285

standard curve (native proteins).

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Intrinsic tryptophan fluorescence of avidin. For tryptophan fluorescence

287

measurements native avidin, avidin-initiator conjugates and avidin-pCBMA conjugates (180

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µL, final concentration of avidin 5 µM) and biotin (20 µL, final concentration 10 µM) were

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mixed in 96-well plate. The tryptophan fluorescence intensities were measured at the

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excitation of 270 nm. The emission spectrum was observed from 300 nm to 400 nm with

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bandwidth of 2 nm using H Synergy Plate reader. The intrinsic fluorescence was measured in

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triplicate.

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Tryptophan fluorescence quenching assay. Intrinsic tryptophan fluorescence

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intensity of native avidin (180 µL, final concentration 5 µM) was measured at the excitation

295

of 270 nm and emission of 300-400 nm in 96-well plate. N-2-bromo-2-methylpropanoyl-β-

296

alanine (free initiator) (20 µL, final concentrations 20 µM, 80 µM, 320 µM, 1.28 mM, 5.12

297

mM) or N-2-methylpropanoyl-β-alanine (free initiation inhibitor) (20 µL, final concentrations

298

20 µM, 80 µM, 320 µM, 1.28 mM, 5.12 mM) were added to native avidin and tryptophan

299

fluorescence was measured again.

300

Biotin effect on quenched fluorescence. Tryptophan fluorescence intensity of native

301

avidin (180 µL, final concentration 5 µM) was measured at the excitation of 270 nm

302

wavelength and the emission was recorded at 300-400 nm. Free initiator (10 µL, final

303

concentrations 5.12 mM) or free initiation inhibitor (10 µL, final concentrations 5.12 mM)

304

were added to avidin solution and florescence intensities were measured. After the

305

fluorescence intensities were recorded with free initiator or free initiation inhibitor, biotin (10

306

µL, final concentration 10 µM) was added to the mixture and fluorescence intensities were

307

measured again. 11 ACS Paragon Plus Environment


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Biotin and biotin-PEG binding kinetics. Kinetic measurements of avidin-pCBMA

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conjugates with biotin and biotin-PEG ligands were taken using a stopped-flow spectrometer

310

with fluorescence detection (Applied Photophysics SX20). The dead time of the instrument

311

was 2 ms. The excitation wavelength was 270 nm with 5 nm bandwidth. Instrument permitted

312

to collect 1000 data points throughput the reaction (0.1-450 s). For all experiments avidin

313

concentration was 0.5 µM (final) and biotin or biotin-PEG concentration was 5.0 µM (final).

314

Reactions were initiated by mixing equal volumes of avidin with its ligands in 0.1 M

315

phosphate buffer (pH 8). Fluorescence was measured in volts. Data were fit to single

316

exponential functions using F(t) = F∞-∆Fexp(-kobst) equation, where, kobs is the observed first-

317

order rate constant, F∞ is the final value of fluorescence and ∆F is the amplitude. In case of

318

native avidin kinetics the data were fit to single exponential functions using F(t) =

319

F∞+∆Fexp(-kobst) equation, where, kobs is the observed first-order rate constant, F∞ is the final

320

value of fluorescence and ∆F is the amplitude. All data analysis was performed in Microsoft

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Excel.

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Biotin-Protein binding kinetics. For biotinylated protein binding kinetics avidin

323

conjugates (0.5 µM final) were mixed with biotin-protein (5.0 µM final) in a stopped-flow

324

accessory on PTI QuantaMaster-400 fluorometer (Horiba Instruments Inc.). The dead time of

325

the instrument was 60 ms. The excitation wavelength was 295 nm with 20 nm bandwidth.

326

Since 270 nm will excite all aromatic residues on proteins, for the analysis of biotin-protein

327

binding kinetics, the excitation wavelength was changed from 270 nm to 295 nm to

328

selectively excite tryptophan residues on avidin only38. Excitation occurred through a 1.96-

329

mm path in the stopped-flow optical cell, and emission was measured through a 7.68-mm path.

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10 data points per second were collected throughout the reaction (15-300 s). Reactions were

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initiated by mixing equal volumes of avidin with its biotinylated ligands in 0.1 M phosphate


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buffer (pH 8). Data were fit to single exponential functions using the same equation used for

333

biotin and biotin-PEG binding kinetics. All data analysis was performed in Microsoft Excel.

334 335

RESULTS AND DISCUSSION

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Avidin conjugate synthesis and characterization. To study the impact of polymer

337

chain length on the permeation of molecules through polymer layers on the surface of

338

bioconjugates, we synthesized avidin-polymer conjugates by growing poly(carboxybetaine

339

methacrylate) (pCBMA) directly from the surface of from avidin. Avidin is a tetrameric

340

protein39, with each monomer containing 10 primary amine groups, (1 α-amine group (N-

341

terminus) and 9 ε-amine groups (lysine residues)). The hydrophilic and zwitterionic polymer,

342

pCBMA, has been shown to have non-fouling properties and thus repel proteins both in vitro

343

and in vivo31. pCBMA has been attached to several proteins without compromising

344

functionality40. Native avidin was modified with an amine-reactive N-2-bromo-2-

345

methylpropanoyl-β-alanine N’-oxysuccinimide bromide ATRP initiator from which a single

346

polymer

347

desorption/ionization time of flight mass spectrometry (MALDI-ToF-MS) showed that we

348

were able to attach the average of 8 initiators per avidin monomer (Figure S1 Supporting

349

Information). PAGE analysis was performed to determine the molecular weight of initiator

350

modified avidin conjugates (Figure S2, Supporting Information).

chains

of

pCBMA

were

grown

(Figure


1a).

Matrix-assisted

laser


351 352 353 354 355 356 357

Figure 1. Synthesis of avidin-pCBMA conjugates using PBPE. a) synthesis of low-density avidinpCBMA conjugates. 1) Single-headed ATRP initiator modification on native avidin, 2) “grafting from” reaction to synthesize avidin-pCBMA conjugates. b) synthesis of high-density avidin-pCBMA conjugates. 1) Double-headed ATRP initiator modification on native avidin, 3) “grafting from” reaction to synthesize double-headed avidin -pCBMA conjugates.

358

The three-dimensional structure of avidin (PDB:2avi) shows that lysines 45, 71 and 111 are

359

located near the biotin binding pocket of avidin, and therefore are ideal modification targets

360

for determining the rate at which ligands can penetrate attached polymers and bind to the

361

surface of the protein (Figure 2a). We have previously demonstrated that trypsin digestion

362

studies followed by mass spectrometry analysis can be used to determine where the ATRP

363

initiators have reacted with proteins41. Trypsin specifically catalyzes the cleavage of peptide

364

bonds at the carboxyl end of positively charged lysine and arginine residues due to the

365

presence of a negatively charged aspartate in the catalytic triad of the trypsin42. Upon

366

modification of a target protein with lysine-reactive ATRP initiators, trypsin is unable to cut

367

the peptide chain at modified lysine residues11,

41.

Trypsin digestion studies with peptide


Page 14 of 30

Page 15 of 30 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

368

mapping using electrospray ionization mass spectrometry (ESI-MS) on the avidin-initiator

369

complexes confirmed that the targeted amino acids (K45, K71 and K111) were covalently

370

modified with ATRP initiator (Figure 2b and Table S1, Supporting Information). Other mass

371

spectrometry methods, such as tandem mass scpectrometry, could have been used for

372

identification of trypsin digested peptide fragments. However, the ESI-MS technique was

373

selected due to its ease of data collection and analysis. After confirming K45, K71 and K111

374

modification, we varied polymerization conditions by changing monomer concentration to

375

synthesize avidin-pCBMA conjugates with four different target lengths or degrees of

376

polymerization (DP): 50, 100, 150 and 200) (Figure 1a).

377 378 379 380 381 382

Figure 2. ESI mass spectroscopy of trypsin digested native avidin and single-headed and doubleheaded initiators modified avidin. a) crystal structure of avidin (PDB:2AVI). K45, K71 and K111 residues located close to the biotin binding site of avidin. b) trypsin digested native avidin. c) trypsin digested avidin-Br. d) trypsin digested avidin-(Br)2. Absence of native peaks for K45 at 1058.1 m/z (GEFTGTYTTAVTATSNEIK m/z, [M+3ACN+2H]2+), K71 at 713.8 m/z (TQPTFGFTVNWK m/z,



Page 16 of 30

383 384 385

[M+2H]2+) and K111 at 639.2 m/z (SSVNDIGDDWK m/z, [M+ACN+2H]2+) suggest that these amine groups were modified with single and double-headed ATRP initiators.

386

Since it is difficult to characterize protein-polymer conjugates as intact conjugates, the

387

polymers were cleaved from the avidin surface using acid hydrolysis. Once cleaved, the

388

polymers were characterized by gel permeation chromotography (GPC) to measure the

389

dispersity and molecular weights of the polymers (Table 1 and Figure S3, Supporting

390

Information). Molecular weights of the conjugates were also estimated using a bicinchonic

391

acid (BCA) assay (Supporting Experimental Section)18. The hydrodynamic diameters (Dh) of

392

avidin conjugates were determined using dynamic light scattering (DLS). As expected, the

393

molecular weight of the conjugates increased linearly with the length of grafted pCBMA. Dh

394

also increased with molecular weight of the attached polymers, and avidin-pCBMA232 Dh was

395

approximately 6-fold over native avidin (Table 1 and Figure S4, Supporting Information).

396

Spectrophotometric assay based on the binding of 4’-hydroxyazobenzene-2-carboxylic acid

397

(HABA) was used to determine the binding activity of avidin conjugates (See Supporting

398

Experimental Section and Table S2, Supporting Information)43. We were now in a position to

399

assess the functionality of each conjugate.

400

Table 1. Characterization of low-density avidin-pCBMA conjugates Sample

Polymerization

D

a)

b)

condition [I] /[M]

h

Avidin-pCBMA

1:100

18.7 ± 1.9

Avidin-pCBMA

1:150

24.8 ± 2.8

Avidin-pCBMA

1:200

Avidin-pCBMA

1:250

0

56

121

170

232

401 402 403 404 405 406 407

0

Estimated conjugate M [kDa] (BCA) 72

d)

c) w

Cleaved polymer M [kDa]; [M /M ] n

w

n

Estimated conjugate M

e) w

12.8 (1.8)

[kDa] (GPC) 120.2

150

27.8 (1.8)

240.2

29.2 ± 3.4

184

39.0 (1.9)

329.8

35.7 ± 2.6

220

53.3 (1.7)

444.2

a)Eight

initiators per avidin monomer, [I]0/[Cu(II)Cl]0/[NaAcs]0[HMTETA]0 = 1:10:12:10; diameters (number distribution) of the avidin-pCBMA conjugates was measured using dynamic light scattering with sample concentration 1.0 mg mL-1 in 100 mM sodium phosphate (pH 8.0) at 25°C; c)Conjugates molecular weight was estimated from BCA as described previously18; d)Number average molecular weight of cleaved pCBMA and dispersity index from GPC; e)Estimated conjugates molecular weight from the measured molecular weight (Mn) of the cleaved polymer (assuming 8 chains of similar length) and initial molecular weight of initiator modified protein. b)Hydrodynamic


Page 17 of 30 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

408 409

Tryptophan Fluorescence changes of avidin upon binding biotin. Prior work has

410

shown that, upon biotin binding, the intrinsic tryptophan fluorescence of avidin is decreased

411

from 337 to 324 nm with a blue shift in emission (Figure S5a, Supporting Information)

412

Surprisingly, when biotin was added to a solution of any avidin-pCBMA conjugates, an

413

increase in tryptophan fluorescence was observed (Figure S5b, Supporting Information).

414

After a series of key experiments (see Figure S5, Supporting Information, and Supporting

415

Discussion), we showed that the increase in intrinsic fluorescence of avidin-pCBMA

416

conjugates after binding biotin was not caused by structural changes upon protein

417

modification, but was a result of quenching of tryptophan fluorescence by the halide-

418

terminated initiator and polymers44. We speculate that the fluorescence quenching of

419

tryptophan residues by bromide group is possibly driven by contact quenching. Tryptophan

420

electron at the excited singlet state is caused to crossover to triplet state by bromide group,

421

and as soon as it crossed to the triplet state it is immediately quenched by either bromide

422

group or oxygen46, 47. We believe that while the bromide group on the ATRP initiator acts as a

423

quencher, causing decreased initial fluorescence, biotin acts as a dequencher and leads to an

424

increase of fluorescence upon binding. This discovery has given us a handle through which,

425

using complex stopped flow fluorescence analysis, we can track the rate at which biotin binds

426

to avidin-pCBMA complexes.

44, 45.

427

Ligand binding rate to avidin through polymer sieves. Most kinetics studies of

428

protein adsorption to polymer-modified surfaces have used surface plasmon resonance (SPR)

429

or quartz crystal microbalance (QCM)48-50. While very sensitive, factors such as polymer

430

density, thickness, viscosity, protein size and difficulties in detection of low molecular weight

431

substrates limit the techniques51,

432

develop an assay to determine the rate of binding of biotinylated ligands to avidin and avidin-

52.

Herein, we used stopped-flow kinetic techniques to



Page 18 of 30

433

pCBMA conjugates. We measured the rate of binding under the first-order reaction conditions

434

where the concentration of biotin was in a molar excess. All the avidin-pCBMA conjugates

435

bound biotin much more slowly (63-73%) than native avidin (Table 2 and Figure S6,

436

Supporting Information). It is worth mentioning that this drop in biotin binding kinetics was

437

not due to avidin conjugates losing their binding activity, but was driven by sieving effect

438

created by covalently attached polymers on avidin surface (Table S2, Supporting Information).

439

We also observed a surprisingly small difference in the rate of biotin binding to the conjugates

440

as a function of polymer chain length (kobs varied from 0.763-0.668 s-1). Thus, we next

441

explored how diffusion of the molecules towards the protein surface changed as a function of

442

the size of the diffusing molecules. We biotinylated a series of proteins of varying sizes:

443

aprotinin (Mw 6.5 kDa), histone (Mw 21.5 kDa) and horse radish peroxidase (HRP, Mw 44.2

444

kDa) (Table S3, Supporting Information).

445 446

Table 2. Biotin, biotin-protein and biotin-PEG binding kinetics to low-density avidin conjugates Sample

Biotin [k/s-1]

Biotinaprotinin [k/s-1]

Biotinhistone [k/s-1]

BiotinHRP [k/s-1]

Native avidin

105.217 ± 12.552

73.451 ± 1.171

15.731 ±1.314

AvidinpCBMA

0.763 ± 0.064

0.565 ± 0.064

AvidinpCBMA

0.739 ± 0.084

AvidinpCBMA AvidinpCBMA

56

121

170

232

447 448 449 450 451 452

5.028 ± 0.433

BiotinPEG 550 Da [k/s-1] 20.933 ± 1.557

BiotinPEG 5 kDa [k/s-1] 17.018 ± 2.001

BiotinPEG 10 kDa [k/s-1] 12.018 ± 0.306

BiotinPEG 30 kDa [k/s-1] 5.892 ± 0.598

0.282 ± 0.017

0.261 ± 0.011

0.738 ± 0.081

0.359 ± 0.033

0.157 ± 0.004

0.156 ± 0.005

0.511 ± 0.057

0.252 ± 0.013

0.218 ± 0.013

0.638 ± 0.077

0.276 ± 0.032

0.159 ± 0.005

0.139 ± 0.004

0.684 ± 0.066

0.493 ± 0.044

0.245 ± 0.011

0.209 ± 0.009

0.622 ± 0.091

0.275 ± 0.022

0.149 ± 0.003

0.136 ± 0.003

0.668 ± 0.078

0.447 ± 0.029

0.231 ± 0.015

0.196 ± 0.008

0.568 ± 0.042

0.266 ± 0.037

0.125 ± 0.005

0.119 ± 0.001

Concentration of avidin was 0.5 µM after mixing and the concentration of biotin and biotin-ligand was 5.0 µM after mixing. Data were fit to single exponential functions using F(t) = F∞-∆Fexp(-kobst) equation, where, kobs is the observed first-order rate constant, F∞ is the final value of fluorescence and ∆F is the amplitude. In case of native avidin kinetics the data were fit to single exponential functions using F(t) = F∞+∆Fexp(-kobst) equation, where, kobs is the observed first-order rate constant, F∞ is the final value of fluorescence and ∆F is the amplitude.

453 18 ACS Paragon Plus Environment

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Biomacromolecules

454

Another criteria in selecting these protein ligands was to keep the number of

455

tryptophan residues to a minimum so that the assays for biotin binding could be performed.

456

Since tryptophan residues are extremely sensitive to their environment53, 54, when bound to

457

avidin the environment of tryptophans on protein ligands will also change leading to changes

458

in emission spectrum (Data not shown). Two of these proteins, aprotinin and histone, do not

459

have tryptophan residues and HRP has only two tryptophan residues that do not interfere with

460

binding emission spectrum (Figure S8, Supporting Information). Using a fluorescamine-based

461

assay, we that were approximately 4 biotin molecules attached per molecule of protein. Since

462

the number of attached biotin molecules was similar amongst the three biotinylated proteins,

463

in the binding kinetics analysis the ratio of avidin to all biotin-protein was normalized to be

464

1:10. We also performed HRP binding kinetics based on biotin number on HRP, instead of

465

HRP-biotin number. It was found that under these conditions the binding of HRP-biotin

466

was 2.6-fold slower (kobs 0.106-0.111) (Figure S9). However, since the binding kinetics were

467

dependent on ligand concentration, it was more accurate to keep the ligand (protein-biotin)

468

higher. The data revealed that the permeation rate of these proteins was sharply dependent on

469

the size of the diffusing protein. The smallest protein ligand aprotinin had the fastest binding

470

rate. A similar trend was observed in a previous study of protein permeation through hydrogel

471

membranes, where the diffusion coefficient of the proteins through a certain mesh sized

472

hydrogels was highly dependent on the protein size55. Interestingly, binding rates of the

473

biotinylated proteins were again barely dependent on the molecular weight of the polymer that

474

had been grown from the surface of avidin. (Table 2). Merrill and colleagues saw similar

475

results in a study of protein adsorption as a function of PEG grafting density, molecular type

476

(linear and star-like), molecular weight and adsorbing protein size56. Merrill’s work

477

demonstrated that covering silicon surfaces with at least half-overlapping PEG chains is

478

important for protein repulsion and that the overlap is independent of PEG molecular weight. 19 ACS Paragon Plus Environment


479

However, to achieve higher overlap and protein repulsion for all PEG molecular weights,

480

higher grafting density was needed. In addition, the amount of a protein adsorbed at a given

481

grafting density and PEG molecular weight correlated with the size of the protein. We

482

therefore sought to determine whether the shape of the ligand and the grafting density of

483

pCBMA on avidin complexes impacted the rate of binding.

484

We selected four PEG polymers of different sizes (biotin-PEG: 550 Da, 5 kDa, 10 kDa

485

and 30 kDa). The binding rate of the biotin-PEG 550 Da to native avidin was 80% slower

486

than biotin itself. The biotin-PEG 550 Da bound to avidin conjugates 3-15% slower than

487

biotin (Table 2). A ten-fold increase in the size of the PEG-biotin chain (biotin-PEG 5 kDa)

488

decreased the rate of binding to avidin by approximately half relative to the rate of binding of

489

biotin, while binding of biotin-PEG 10 kDa was 78-79% slower than biotin. Lastly, the

490

binding rate of biotin-PEG 30 kDa to avidin was 80-82% slower than that of biotin. In all

491

cases, we observed that larger biotin-PEG ligands bound slower to the active site. We again

492

did not observe a pronounced dependence of permeation rate of linear PEGs on the pCBMA

493

molecular weight that was attached to avidin (Table 2). We measured hydrodynamic

494

diameters of both biotin-protein and biotin-PEG ligands using DLS and found that the

495

smallest ligand was PEG 550 Da, followed by aprotinin, PEG 5K, histone, HRP, PEG 10K

496

and finally PEG 30K (Table S3 and S4, Supporting Information). After analyzing both the Dh

497

and permeation rates, we noticed that the permeation rate for biotinylated aprotinin (Dh 2.4

498

nm) was slower than the permeation rate of biotin-PEG 550 Da (Dh 1.9 nm) (Figure 3a).

499

These observations led us to hypothesize that for single-headed initiator “grown from” avidin

500

conjugates, it was the ligand size, not shape, that was important in determining the permeation

501

rate through polymer shell. These data suggested that increasing polymer density around the

502

active site of avidin may drive more effective sieving. Unfortunately, until now the number of

503

chains grown from a protein by ATRP has been directly proportional tot he number of amine20 ACS Paragon Plus Environment

Page 20 of 30

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Biomacromolecules

504

reactive NHS-functionalized ATRP initiators per protein molecule. We were interested in

505

whether we could overcome this limitation by growing mutliple polymer chains from single

506

sites.

507 508 509 510 511 512 513 514 515 516

Figure 3. Binding rates of biotinylated ligands to avidin-pCBMA conjugates as a function of ligand size. a) binding rates of biotin ligands to low-density avidin-pCBMA conjugates. Avidin-pCBMA56 (dark magenta), avidin-pCBMA121 (purple), avidin-pCBMA170 (deep pink), and avidin-pCBMA232 (pink). b) binding rates of biotin ligands to high-density avidin-pCBMA conjugates. High-density avidin-pCBMA58 (dark blue), high-density avidin-pCBMA109 (blue), high-density avidin-pCBMA152 (cyan), and high-density avidin-pCBMA182 (light violet). Ligands: biotin-PEG 550 Da – 1.9 ± 0.8 nm, biotin-aprotinin – 2.4 ± 0.4 nm, biotin-PEG 5K – 4.2 ± 0.5 nm, biotin-histone – 4.6 ± 0.2 nm, biotinHRP – 5.1 ± 0.7 nm, biotin-PEG 10K – 6.1 ± 0.5 nm and biotin-PEG 30K – 9.4 ± 0.9 nm.

517

Synthesis and characterization of a high-density conjugates. To increase the

518

polymer density, we synthesized a novel double-headed ATRP initiator that allowed us to

519

grow two polymer chains from each initiation site. First, polymerization was performed from

520

unattached double-headed ATRP initiator to confirm the growth of both polymer chains from

521

one initiator and to optimize the conditions for conjugate synthesis (Figure S10, Supporting

522

Information). GPC before and after acid hydrolysis of the synthesized polymers was used to

523

prove that one double-headed initiator led to the growth of two polymer chains in solution

524

(Table S5, Supporting Information).

525

We next reacted the double-headed ATRP initiator with primary amines on the surface

526

of avidin (Figure 1b) and used MALDI-ToF to show that there were an average of 7 double-

527

headed initiators on each avidin monomer (Figure S11, Supporting Information). 21 ACS Paragon Plus Environment


528

Unsurprisingly, the larger initiator lost its ability to react with at least one lysine previously

529

accessible by the single-headed initiator. While the degree of modification decreased in the

530

case of double-headed initiator, the number of polymer chains grafted from the avidin surface

531

was still higher. The initiation sites that the double-headed initiator targeted were well-

532

distributed on the avidin surface and a polymer density of one polymer per 0.5 chains nm-2 for

533

the double-headed initiator “grown from” conjugates was calculated. This led to a 1.8-fold

534

higher pCBMA grafting density as compared to the single-headed initiator derived conjugates

535

(0.29 polymer chains nm-2), i.e. the grafting density incresed by 75%. Trypsin digestion

536

followed by ESI-MS was again used to determine that K45, K71 and K111 near the binding

537

site had still reacted with double-headed ATRP initiator (Figure 3d and Table S1, Supporting

538

Information). We then characterized the molecular weight, degree of polymerization and the

539

hydrodynamic sizes of the high-density avidin-pCBMA conjugates. As expected, the

540

molecular weights and hydrodynamic sizes of the high-density conjugates were much larger

541

than those for the low-density conjugates for the same degree of polymerization. Next, GPC

542

was used for determination of cleaved polymer molecular weight and dispersity (Table 3 and

543

Figure S12, Supporting Information). It is worth mentioning that both low and high-density

544

conjugates had molecular weight dispersities at the higher end of ATRP approaches. However,

545

since all conjugates (low and high-density with different polymer lengths) had similar

546

dispersities, ligand binding kinetics results could be compared within the conjugate families.

547

DLS was used for hydrodynamic diameter measurements (Table 3 and Figure S13,

548

Supporting Information). Spectrophotometric method was used to determine HABA binding

549

activity of high-density avidin conjugates (Table S2, Supporting Information).

550 551 552 22 ACS Paragon Plus Environment

Page 22 of 30

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553

Biomacromolecules

Table 3. Characterization of high-density avidin-pCBMA conjugates Sample

Polymerization condition [I] /[M]

a)

D

b)

h 7


d)

c) w

Avidin-pCBMA58

1:100

25.1 ± 1.7

[kDa] (BCA) 124

Avidin-pCBMA109

1:150

30.8 ± 4.2

Avidin-pCBMA152

1:200

Avidin-pCBMA182

1:250

0

Cleaved polymer M [kDa]; [M /M ] n

w

n


e) w

13.4 (1.6)

[kDa] (GPC) 206.6

202

25.1 (1.8)

370.4

34.4 ± 3.8

290

34.9 (1.8)

507.6

38.8 ± 1.2

408

41.8 (1.8)

604.2

0

554 555 556 557 558 559 560 561 562

initiators per avidin monomer, [I]0/[Cu(II)Cl]0/[NaAcs]0[HMTETA]0 = 1:10:1.2:10; diameters (number distribution) of the avidin-pCBMA conjugates was measured using dynamic light scattering with sample concentration 1.0 mg mL-1 in 100 mM sodium phosphate (pH 8.0) at 25°C; c)Conjugates molecular weight was estimated from BCA as described previously18; d)Number average molecular weight of cleaved pCBMA and dispersity index from GPC; e) Estimated conjugates molecular weight from the measured molecular weight (Mn) of the cleaved polymer (assuming 14 chains of similar length) and initial molecular weight of initiator modified protein.

563

Polymer grafting density effect on binding kinetics. High-density avidin-pCBMA

564

conjugates had a two-fold decrease in biotin binding rate compared to low-density conjugates

565

(Table 4) (kobs differed from 0.345-0.299 s-1 versus 0.763-0.668 s-1, respectively). Doubling

566

the grafting density from each initiation site on avidin had a marked effect on the binding rate

567

of biotinylated macromolecules. As expected, aprotinin had the fastest permeation rate,

568

followed by histone and then HRP. The permeation rate of these biotinylated proteins through

569

the high-density polymers shell on avidin to the binding site was approximately ten-fold lower

570

than that for the low-density avidin-pCBMA conjugates. We were surprised to see that high-

571

density avidin-pCBMA conjugates still bound the largest protein ligand (HRP), although with

572

a decreased rate. This is consistent with a previous theoretical study, which postulated that

573

just covering the surface with PEG polymers was not sufficient to prevent the proteins from

574

reaching the surface57. The study revealed that proteins can permeate through polymer layers

575

and localize between polymer chains. We observed a strong dependence of biotin-protein

576

binding on the pCBMA grafting density. The rates of binding for the high-density avidin-

577

pCBMA conjugates were also not strongly impacted by the molecular weight of the grafted

a)Seven

b)Hydrodynamic



Page 24 of 30

578

pCBMA (Table 4). De Gennes and colleagues saw similar results in a theoretical

579

characterization of protein resistance properties of PEG chains attached to hydrophobic

580

surfaces, by calculating the steric repulsion free energy initiated by protein compressing PEG

581

chains and hydrophobic interaction free energies as a function of polymer grafting density and

582

molecular weight58. They found that higher grafting density exhibits stronger protein

583

repulsion due to the compression of PEG chains, and thus is more important than polymer

584

molecular weight in preventing protein adsorption.

585 586 587

Table 4. Biotin, biotin-protein and biotin-PEG binding kinetics to high-density avidin conjugates Sample

Biotin [k/s-1]

Biotinaprotinin [k/s-1]

Biotinhistone [k/s-1]

BiotinHRP [k/s-1]

Native avidin

105.217 ± 12.552

73.451 ± 1.171

15.731 ±1.314

AvidinpCBMA

0.345 ± 0.032

0.056 ± 0.005

AvidinpCBMA

0.331 ± 0.024

AvidinpCBMA AvidinpCBMA

58

109

152

182

588 589 590 591

5.028 ± 0.433

BiotinPEG 550 Da [k/s-1] 20.933 ± 1.557

BiotinPEG 5 kDa [k/s-1] 17.018 ± 2.001

BiotinPEG 10 kDa [k/s-1] 12.018 ± 0.306

BiotinPEG 30 kDa [k/s-1] 5.892 ± 0.598

0.039 ± 0.004

0.028 ± 0.001

0.082 ± 0.003

0.046 ± 0.004

0.025 ± 0.001

0.018 ± 0.002

0.051 ± 0.007

0.035 ± 0.002

0.026 ± 0.001

0.076 ± 0.002

0.044 ± 0.003

0.026 ± 0.002

0.015 ± 0.001

0.309 ± 0.035

0.047 ± 0.003

0.034 ± 0.001

0.025 ± 0.001

0.073 ± 0.003

0.042 ± 0.002

0.025 ± 0.001

0.016 ± 0.001

0.299 ± 0.017

0.040 ± 0.001

0.032 ± 0.002

0.025 ± 0.001

0.067 ± 0.002

0.041 ± 0.002

0.022 ± 0.002

0.016 ± 0.001

Concentration of avidin was 0.5 µM after mixing and the concentration of biotin and biotin-ligand was 5.0 µM after mixing. Data were fit to single exponential functions using F(t) = F∞-∆Fexp(-kobst) equation, where, kobs is the observed first-order rate constant, F∞ is the final value of fluorescence and ∆F is the amplitude.

592 593

The binding kinetics for the interaction between high-density avidin-pCBMA

594

conjugates and biotinylated-PEG ligands had a sharp dependence on the size of the PEG

595

(Table 4 and Figure 3b). The binding rates of all molecules to these conjugates were ten-fold

596

slower than for the low-density conjugates. Again, we found that molecule shape was


Page 25 of 30 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

597

unimportant in diffusion through grafted polymers, and diffusion rate changed as a function of

598

ligand size.

599 600

CONCLUSIONS

601

For the first time, we have quantified the rates of binding of globular and linear

602

macromolecules to a protein surface through a layer of covalently attached polymers. Surface

603

initiated ATRP was used to synthesize avidin-pCBMA bioconjugates that were used to

604

investigate the role of polymers molecular weight and grafting density in shielding protein

605

surface from molecule penetration. Stopped-flow kinetics proved to be a powerful tool in

606

measuring the binding rates of biotinylated molecules of varying shape and size to the avidin

607

binding pocket through grafted pCBMA polymers. We have created a unique double-headed

608

ATRP initiator which enabled us to synthesize protein-polymer bioconjugates with high

609

grafting densities, without the need to change the protein itself. This chemistry may provide

610

new avenues in creation of bioconjugates covered with dense polymer shells using double,

611

triple or even multi-headed initiators. We have concluded that there appears to be no specific

612

pCBMA molecular weight that is necessary to affect the ligand binding rate, at least in the

613

range of ligand sizes and pCBMA lengths we studied. Instead for a given pCBMA molecular

614

weight, the grafting density slowed the diffusion and binding of ligands to the protein active

615

site.

616

independent of the ligand shape. We are now exploring molecule diffusion rates through

617

dendritic and cross-linked polymers grown from a protein surface.

Additionally, we discovered that molecule binding rate depends on ligand size,

618 619

Supporting Information. Synthesis of double-headed ATRP initiator, discussion of intrinsic

620

tryptophan fluorescence changes of avidin conjugates upon biotin binding, MALDI-ToF data,

621

SDS-PAGE data, GPC traces of cleaved polymers, DLS data, tryptophan fluorescence data, 25 ACS Paragon Plus Environment


622

peak analysis of peptide fragments after tryosin digestion for native avidin, HABA binding

623

activity of avidin-pCBMA conjuagtes, characterizations of biotin-PEG and biotin-protein

624

substrates, characterizations of pCBMA polymers grown from single-headed and double-

625

headed ATRP initiators.

626

AUTHOR INFORMATION

627

Corresponding Author

628

*E-mail: [email protected]

629

Author Contributions

630

B.K. synthesized and characterized avidin-pCBMA conjugates, biotinylated proteins,

631

performed trypsin digestion and mass spectrometer analysis experiments, measured binding

632

kinetics, performed tryptophan fluorescence assays and drafted the manuscript. H.M.

633

synthesized and characterized ATRP initiators, performed free polymerization from single

634

and double-headed initiators, characterized polymers and helped to design experiments, A.L.

635

was involved in experimental design and result discussions, K.M., J.S.M. and A.J.R.

636

supervised the project and provided guidance. The manuscript was written through

637

contributions of all authors. All authors have given approval to the final version of the

638

manuscript.

639

Acknowledgements

640

The authors would also like to thank Weihang Ji (Carnegie Mellon University) for performing

641

PAGE analysis, Tina Lee and David Hackney (Carnegie Mellon University, Pittsburgh, PA)

642

for sharing equipment, Gordon Rule, Alan Waggoner and Frederick Lanni (Carnegie Mellon

643

University, Pittsburgh, PA) for discussion about tryptophan fluorescence assays and James

644

Winsor (Carnegie Mellon University) for assistance in stopped-flow kinetics experiments.

645

Funding


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Biomacromolecules

646

The authors acknowledge financial support provided by Carnegie Mellon University Center

647

for Polymer-Based Protein Engineering and DTRA grant: HDTRA1-18-1-0028 Carnegie

648

Mellon FRBAA14-BR-TA7-G19-2-0124.

649

Notes

650

The authors declare no competing financial interest.

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Molecular Sieving on the Surface of a Nano-Armored Protein

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