Modulation of the Binding Affinity of Polyzwitterion-Conjugated Protein

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Modulation of the Binding Affinity of Polyzwitterion-Conjugated Protein by Ion-Specific Effects in Crowded Environments Wangqin Song, Jie Zhu, Lvdan Liu, and Guangming Liu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b04314 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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The Journal of Physical Chemistry

Modulation of the Binding Affinity of Polyzwitterion-Conjugated Protein by Ion-Specific Effects in Crowded Environments

Wangqin Song,† Jie Zhu,† Lvdan Liu, Guangming Liu*

Department of Chemical Physics, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, P. R. China 230026

1

ACS Paragon Plus Environment


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Abstract. Macromolecular crowding could influence the binding affinity of the polyzwitterion-conjugated

proteins.

Herein,

the

hydrolysis

of

N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide by the poly(carboxybetaine) conjugated α-chymotrypsin (PCT) was employed as a model system to investigate the modulation of the binding affinity of the polyzwitterion-conjugated proteins by the ion-specific effects in the crowded environments. In comparison with the bare α-chymotrypsin (BCT), the binding affinity of the PCT to the peptide is stronger in the dilute solutions but becomes weaker in the crowded environments. Our study demonstrates that the kosmotropic Ac- anion is incapable of achieving a stronger enzymatic binding affinity of the PCT than the BCT in the highly crowded environments. By contrast, the binding affinity of the PCT can be enhanced to be stronger than that of the BCT by the chaotropic SCN- anion in the crowded environments.

*To whom correspondence should be addressed. Email: [email protected].

2


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Introduction Development of therapeutic proteins has significantly changed the situation of treatment of many diseases.1-4 Unfortunately, the native therapeutic proteins are often accompanied with poor pharmacokinetic properties due to the low stability and rapid clearance by organism, which ultimately reduces their therapeutic efficacy.5 To increase the stability and circulation time of the therapeutic proteins, the strategy of PEGylation has been established through the conjugation of biocompatible poly(ethylene glycol) (PEG) chains to the proteins.5-7 However, the PEGylation would generally lead to a reduction in biological activity of the protein drugs owing to the decrease of protein binding affinity.8,9 Recently, poly(carboxybetaine) (PCB), a kind of polyzwitterion, was introduced as an alternative biocompatible polymer to form polyzwitterionic protein conjugates, which could improve the stability without sacrificing the binding affinity or bioactivity of the proteins in the dilute solutions.10 On the other hand, the therapeutic proteins need to be delivered into cell to exert their biological actions inside cytoplasm because many therapeutic targets are located inside cells.11-13 The intracellular environment contains not only the high concentration of macromolecular crowders but also various small ions.14,15 The concentration of macromolecular crowders in cytoplasm is up to 300-400 mg mL-1 and the concentration of small ions in the intracellular fluid is ~ 0.15 M.16,17 Previous studies have shown that the macromolecular crowding could influence the enzyme-substrate

affinity

by

increasing

diffusion

resistance.18,19

For

the

polymer-conjugated proteins, both the steric hindrance generated by the grafted 3



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polymer chains and the local viscosity around the binding site could be altered by the crowded environments, which would also affect the binding affinity of the proteins. At the same time, it is anticipated that the binding affinity of the polymer-conjugated proteins could also be modulated by the small ions in the cellular environment via ion-specific interactions with either the proteins or the grafted polymer chains.20 In this work, the hydrolysis of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) by the PCB conjugated α-chymotrypsin (PCT) was employed as a model system to investigate how the binding affinity of the polyzwitterion-conjugated proteins can be modulated by the ion-specific effects in the crowded environments.

Experimental Section Materials. α-chymotrypsin (CT) from bovine pancreas, lyophilized powder, was purchased from TCI. N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, 98%)

was

purchased

from

2-bromo-2-methylpropionyl

Hwrk

bromide

Chemical (98%)

and

was

used

purchased

as

received.

from

Alfa.

β-Propiolactone (99%) was purchased from Macklin. N-[3-(Dimethylamino)propyl] methacrylamide (DMAPMA, 97%) was purchased from Sigma-Aldrich and filtered through

a

basic

alumina

column

to

remove

the

radical

inhibitor.

1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA, 97%) was purchased from Energy Chemical and used as received. Copper (II) bromide (CuBr2, 99%) was purchased from Sinopharm and used as received. Copper(I) bromide (CuBr) was 4


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prepared from CuBr2 by reacting with sodium sulfite. Dextran (~ 70 kDa), N-hydroxysuccinimide (NHS, 98%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 98.5%) and all the salts (99.99%, metals basis) were purchased from Aladdin and used as received. The water used was purified by filtration through a Millipore Gradient system after pre-distillation, giving a resistivity of 18.2 MΩ cm.

Characterizations. All proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AV400 NMR spectrometer. Mass spectra were recorded on a MALDI-TOF Autoflex III SmartBeam mass spectrometer (Bruker Daltonics). The number-average molecular mass (Mn) of the polyzwitterion-protein conjugate was determined by gel permeation chromatograph (GPC) (Waters 1515) using monodisperse poly(ethylene glycol) as the standard and salt solution (1 M NH4NO3) as the eluent with a flow rate of 1.0 mL min−1. The enzymatic reaction progress was followed with a UNICO 2802PCS UV/visible spectrophotometer. The hydrodynamic radius of the polyzwitterion-conjugated CT was determined by using a commercial laser light scattering spectrometer (ALV/DLS/SLS-5022F) at a scattering angle of 90°. The measurements of circular dichroism (CD) spectra were conducted on a Chirascan qCD Circular Dichroism Spectrometer.

Synthesis of NHS-functionalized ATRP initiator and preparation of the CT-initiator

conjugate.

The

NHS-functionalized

atom

transfer

radical

polymerization (ATRP) initiator was synthesized according to the previous study.21 The successful synthesis of the NHS-functionalized ATRP initiator was confirmed by 5



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the 1H NMR measurement (Figure S1, Supporting Information). The CT-initiator conjugate was prepared by adding the NHS-functionalized ATRP initiator to a phosphate buffer (~ pH 8.0) containing CT. The details for the preparation of the CT-initiator conjugate can be found elsewhere.21 The successful preparation of the CT-initiator conjugate was confirmed by the 1H NMR measurement (Figure S2, Supporting Information).21

About ten of the ATRP initiators were grafted on the

surface of CT, which was estimated from the MALDI-TOF MASS spectra (Figure S3, Supporting Information).

Preparation of polyzwitterion-conjugated CT. The zwitterionic monomer was synthesized according to the procedure reported in the previous work.22 The successful synthesis of the zwitterionic monomer was confirmed by the 1H NMR measurement (Figure S4, Supporting Information). The polyzwitterion-conjugated CT was prepared by using the surface-initiated ATRP (SI-ATRP) method (Figure 1). Briefly, a 30 mL of phosphate buffer solution containing the zwitterionic monomer (2.0 mmol) and the CT-initiator conjugate (100 mg, 3.6 μmol) was sealed and bubbled with nitrogen in an ice bath for ~ 60 min. A 10 mL of deoxygenated phosphate buffer solution containing HMTETA (24 μL, 0.09 mmol) and Cu(I)Br (13 mg, 0.09 mmol) was then added to the above solution under the protection of nitrogen. The mixture was sealed and stirred for ~ 8 h at 4 °C. Afterwards, the polyzwitterion-conjugated CT was obtained by dialysis in water and then lyophilized. The Mn of the prepared polyzwitterion-conjugated CT was ~ 1.3 × 105 g mol-1 determined by GPC.

6


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Figure 1. Schematic illustration of the preparation of the polyzwitterion-conjugated CT from the CT-initiator conjugate by using the SI-ATRP method.

Hydrolysis of Suc-AAPF-pNA. All the hydrolysis reactions were carried out in Tris-HCl buffer (~ pH 8.0) at 25 °C. The concentration of bare CT (BCT) or PCT in the reaction mixture was fixed at 2 µM with 0.1 mg/mL BSA as a blocking agent. Suc-AAPF-pNA was added to the reaction mixture at varying concentrations from 0 to 1.44 mM. The concentration of dextran varied from 0 to 200 mg/mL. The reaction was initiated by adding the BCT or PCT to a Tris-HCl buffer containing substrate, salt and dextran. The reaction progress was followed by monitoring the time dependent absorbance of the released p-nitroanilide at 412 nm. The ionic strength in the reaction mixture was fixed at 0.15 M for the salt solutions. The salt solutions were prepared by adding the relevant salts (100 mM) to the Tris-HCl buffer (50 mM). Stability of enzyme. The 20 µM of BCT or PCT was incubated in the Tris-HCl buffer with 0.1 mg/mL BSA as a blocking agent at temperatures of 25, 40, 45, 50 and 55 °C. 7



After 10 min of incubation at each temperature, the enzyme was diluted into the Tris-HCl buffer containing substrate, salt, and dextran to a concentration of 0.2 µM at 25 °C. The activity of the corresponding enzyme derived from the initial velocity was measured by the hydrolysis of the Suc-AAPF-pNA with a concentration of 1.26 mM.

Results and Discussion The hydrolysis of Suc-AAPF-pNA by α-chymotrypsin releases the product of p-nitroanilide.19 In Figure 2a, the reaction progress of the hydrolysis of the peptide by the BCT is followed by monitoring the time dependent absorbance of the released p-nitroanilide at 412 nm in a dilute and salt-free solution. Here, we choose the initial velocity (v0), which is defined as the slope of the line fitting of the first 30 s of the absorbance/time data, as the reaction rate.19 Consequently, the Michaelis constant (KM) can be obtained by curve fitting of the experimental data using the equation v0 = vmax[S]/(KM+[S]) as shown in Figure 2b, where [S] is the substrate concentration and vmax is the maximum velocity. 0.4

(a)

12.0 -1

0.3



v s

Abs nm

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0.2 0.1 0.0

(b)

8.0

4.0

0.0 0

15

30

45

60

0.0

Time / s

0.5

-3

1.0

1.5

[S] / 10 M

Figure 2. The hydrolysis of Suc-AAPF-pNA by the BCT in dilute and salt-free 8


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solutions. (a) Time dependence of absorbance (Abs) of the p-nitroanilide at the wavelength (λ) of 412 nm with the substrate concentration of 180 μM. (b) The reaction rate (v0) as a function of the substrate concentration ([S]). The dashed line is the curve fit to the data.

The hydrolysis of Suc-AAPF-pNA by α-chymotrypsin can be described by the following equation:19,23

(1) The KM is defined as (k-1+kcat)/k1, which can be assumed to reflect the binding affinity between the enzyme (E) and the substrate (S) to form the enzyme-substrate complex (ES) under a certain approximation. Here, k1, k−1, kcat, and P are the forward rate constant, reverse rate constant, catalytic rate constant, and product, respectively. For the enzyme reaction employed here, the KM is used to evaluate the affinity between the BCT or PCT and the substrate according to the previous studies.10,24,25 Figure 3a shows that KM increases with increasing concentration of dextran (Cdextran) for both the BCT and the PCT, indicating that the binding affinity between the enzyme and the substrate is decreased with increasing crowding due to the increased diffusion resistance generated by the crowding agents. As the size of the substrate is much smaller than the BCT and PCT, it is likely that the influence of crowding on the binding affinity should be dominated by the decrease of diffusion of the substrate. In addition, similar to the previous study,19 the v0 also decreases with increasing crowding (Figure S5, Supporting Information). 9



PCT BCT

2.4 -3

KM / 10 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

salt-free

(a)

1.6 0.8 0.0

50

0

C

100

dextran

150

200

/ (mg/mL)

Figure 3. (a) KM for the hydrolysis of Suc-AAPF-pNA by either the BCT or the PCT as a function of the concentration of dextran (Cdextran) in the salt-free solutions. (b) Schematic illustration of the BCT and PCT in the crowded environments. The PCT is physically larger than the BCT and therefore its diffusion resistance is higher in a crowded environment. For clarity, the relative sizes of BCT, PCT, substrate, and crowder do not exactly reflect the real difference in size between them.

In the salt-free solution in the absence of crowding agents, the KM of PCT is lower than that of BCT, implying that the binding affinity of PCT to the peptide is stronger than that of BCT. This is because the hydrophobic interaction between the binding site of enzyme and the substrate is enhanced via grafting the superhydrophilic polyzwitterionic chains on the protein surface.10 However, the value of KM of the PCT becomes higher than that of the BCT when dextran is added. This means that the PCT exhibits a weaker binding affinity than the BCT in the crowded environments. Furthermore, the difference in KM between the PCT and the BCT becomes larger with increasing Cdextran, indicating that the increasing crowding has a larger influence on 10


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the enzymatic binding affinity of the PCT than that of the BCT. The PCT was prepared by grafting about ten of polyzwitterionic chains on the protein surface using the SI-ATRP method (Figure 1). The PCT has a Mn of ~ 1.3 × 105 g mol-1 with a hydrodynamic radius of ~ 20 nm, whereas the BCT has a molecular weight of ~ 2.6 × 104 g mol-1 with a size of ~ 4 nm.26 Therefore, the grafting of polyzwitterionic chains to the protein surface renders the PCT a larger size and a higher diffusion resistance than the BCT. This may lead to a weaker enzymatic binding affinity of the PCT than the BCT in the crowded environments (Figure 3b). Besides, the grafted polyzwitterionic chains of the PCT could adopt a more compacted conformation in the crowded environments compared with that in the dilute solution, which would lead to the increases of both the steric hindrance and the local viscosity around the binding site of the PCT. This may also contribute to the lower binding affinity of the PCT than the BCT in the crowded environments. In fact, the intracellular environment also contains many types of ions besides the high concentration of crowding agents.27 Therefore, it is expected that the enzymatic binding affinity could be modulated by the ion-specific effects. We chose three types of typical anions from the Hofmeister series, i.e., acetate (Ac-), chloride (Cl-) and thiocyanate (SCN-), to explore how the enzymatic binding affinity can be modulated by the ion-specific effects in the crowded environments. SCN- is a chaotropic anion, whereas CH3COO- is a kosmotropic anion.17,28,29 Cl- locates at the border between chaotropes and kosmotropes in the Hofmeister series.17,28,29 In Figure 4a and 4b, the KM for both the BCT and the PCT decreases following the series Cl- > salt-free > Ac- > 11



SCN- regardless of the extent of crowding. This fact indicates that the binding affinity of the enzyme increases following the series Cl- < salt-free < Ac- < SCN-.

2.1

-3

1.4

BCT

-

(a)

2.4 -3

Cl salt-free Ac SCN

KM / 10 M

-

KM / 10 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

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0.7 0.0

0

50

100

150

Cdextran / (mg/mL)

1.6

PCT

50

100

(b)

0.8 0.0

200

Cl salt-free Ac SCN

0

150

Cdextran / (mg/mL)

200

Figure 4. Enzymatic binding affinity of the BCT and the PCT measured as Michaelis constant KM as a function of the concentration of dextran (Cdextran) in the presence of different types of anions with Na+ as the common cation. (a) The KM of the BCT. (b) The KM of the PCT. Here, the ionic strength is fixed at 0.15 M for the salt solutions.

For both the BCT and the PCT, the KM in the presence of Ac- is smaller than that in the salt-free solution, indicating that the enzymatic binding affinity becomes stronger with the addition of Ac-. This is because the hydrophobic interaction between the enzyme and the substrate could be strengthened in the presence of strongly hydrated kosmotropic anions via the salting-out effects.30 Such an increase in hydrophobic interaction is effective for both the BCT and the PCT. More specifically, the competition for the water molecules in the hydration layer of either the enzyme molecules or the grafted polyzwitterionic chains by Ac- could lead to the amplification of the hydrophobic interaction between the BCT or PCT and the substrate.30 12


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In the presence of SCN-, the enzymatic binding affinity is also increased in comparison with that in the salt-free solution and is even stronger than that in the presence of Ac-. It is generally thought that SCN- is a weakly hydrated chaotropic anion, which should reduce the hydrophobic interaction via the salting-in effects.30 Thus, the mechanism of the increased enzymatic binding affinity for SCN- should be different from that for Ac-. The isoelectric point of α-chymotrypsin is ~ 8.8.31 As the pH of the prepared buffer solution used here is ~ 8.0, the enzyme molecules are positively charged under the experimental conditions in this work. According to the Collins’ law of matching water affinities,32 the interaction strength between the anions and the weakly hydrated amine groups should increase following the order Ac- < Cl- < SCN-, since the anionic hydration strength increases following the reverse order.17,28,29,33,34 Thus, the weakly hydrated SCN- anions would form strong ion pairs with the weakly hydrated amine groups.32 As a result, SCN- could effectively neutralize the positive charges of either the enzyme molecules or the conjugated polyzwitterionic chains through the formation of ion pairs, thereby strengthening the hydrophobic interaction between the enzyme and the substrate. As Cl- locates at the border between chaotropes and kosmotropes in the Hofmeister series, it is unlikely that Cl- could increase the enzymatic binding affinity through either the salting-out effects like the Ac- or the ion-pairing interactions like the SCN-. In fact, the binding affinity of both the BCT and the PCT is slightly weakened with the addition of Cl- compared with that in the salt-free solution. This may be attributed to a slight disruption of enzyme structure by the addition of NaCl.35 13



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In the presence of Ac-, the KM of the PCT is lower than that of the BCT when the concentration of dextran is ≤ 50 mg mL-1 but is higher than that of the BCT when the concentration of dextran is ≥ 100 mg mL-1 (Figure 5a), indicating that the binding affinity of the PCT is weaker than that of the BCT in the highly crowded environments in the presence of kosmotropic Ac- anions. On the other hand, Figure 5b shows that the difference in KM (ΔKM) between the BCT and the PCT in the presence of Ac- is smaller than that in the salt-free solutions in the highly crowded conditions. These facts suggest that Ac- is incapable of achieving a stronger enzymatic binding affinity of the PCT than the BCT in the highly crowded environments, but it can reduce the difference in binding affinity between the PCT and the BCT in the highly crowded conditions compared with that in the salt-free solutions. Thus, the salting-out effects exerted by the Ac- anions can more effectively increase the enzyme-substrate affinity for the PCT than the BCT in the crowded environments. This could be induced by the cooperative effects between the superhydrophilic polyzwitterionic chains and the strongly hydrated Ac- anions on the increase of the hydrophobic interaction between the enzyme and the substrate. Figure 5c shows that the KM of the PCT is lower than that of the BCT from the dilute solution to the crowded environments in the presence of SCN-. This result implies that the SCN- has an ability to achieve a stronger binding affinity of the PCT than that of the BCT in the crowded environments. Thus, the situation that the PCT has a weaker binding affinity than the BCT in the salt-free and crowded environments is reversed when the SCN- anions are added to the solutions (Figure 5d). 14


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Ac

-

(a)

M(BCT-PCT)

2.0

K

1.0

2.0

50

PCT BCT

SCN

-

-0.3 -0.6 0

(c)

M(BCT-PCT)

1.0

K

0.5 0.0

0.0

200

-3

1.5

150

100

Cdextran / (mg/mL)

0

50

Ac

0.3

0.6

100

150

Cdextran / (mg/mL)

200

50

100

150

Cdextran / (mg/mL)

200

(d)

salt-free -

-3

0

(b)

salt-free -

/ 10 M

0.0

0.6

-3

PCT BCT

-3

KM / 10 M

3.0

KM / 10 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


/ 10 M

Page 15 of 25

SCN

0.3 0.0 -0.3 -0.6 0

50

100

150

Cdextran / (mg/mL)

200

Figure 5. (a) KM for the hydrolysis of the Suc-AAPF-pNA by either the BCT or the PCT as a function of the concentration of dextran (Cdextran) in the presence of Ac-. (b) The difference in KM (ΔKM) between the BCT and the PCT as a function of the concentration of dextran (Cdextran) in the salt-free solutions and in the NaAc solutions. (c) KM for the hydrolysis of the Suc-AAPF-pNA by either the BCT or the PCT as a function of the concentration of dextran (Cdextran) in the presence of SCN-. (d) The difference in KM (ΔKM) between the BCT and the PCT as a function of the concentration of dextran (Cdextran) in the salt-free solutions and in the NaSCN solutions.

As discussed above, the ion-pairing interactions between the SCN- anions and the positively charged amine groups would increase the hydrophobic interaction between the enzyme and the substrate, giving rise to an increase in the enzymatic binding 15



affinity. Nevertheless, the ion-pairing interactions could also lead to a competition for the active site accompanied by a denaturation of the enzyme (Figure 6a).36,37 Figure 6b demonstrates that the binding of SCN- could induce a change of the secondary structure of the BCT, as reflected by the shift of the negative band at ~ 203 nm of the CD spectrum in the presence of SCN- compared with that in the absence of SCN-. This result suggests the change of conformation of the BCT accompanied by a decrease of protein stability induced by the binding of SCN- through the ion-pairing interactions. As a consequence, a decrease in enzymatic activity of the BCT is resulted (Figures S6 and S7, Supporting Information). Thus, the binding affinity of the BCT in the presence of SCN- is determined by the competition between the two

Ellipticity / mdeg

opposing factors mentioned above.

(b)

Tris-HCl Tris-HCl+NaSCN

10 0 -10

BCT

-20 200

220

240

260

Wavelength / nm

Ellipticity / mdeg

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 16 of 25

(d)

Tris-HCl Tris-HCl+NaSCN

10 0 -10

PCT

-20 200

220

240

Wavelength / nm 16


260

Page 17 of 25

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Figure 6. (a) Schematic illustration of the modulation of binding affinity of the BCT by SCN- in the crowded environments. The cross symbol indicates that the interactions between the enzyme active site and the substrate are weakened due to the competitive binding of SCN- to the active site. For clarity, the Na+ cations are not depicted here. (b) Far-UV CD spectra of the BCT (10 μM) in the presence and absence of NaSCN (1 mM) in the Tris-HCl buffer (5 mM). (c) Schematic illustration of the modulation of binding affinity of the PCT by SCN- in the crowded environments. The tick symbol indicates that the interactions between the enzyme active site and the substrate are almost uninfluenced by the addition of SCN- due to the protection of the grafted polyzwitterionic chains. For clarity, the Na+ cations are not depicted here. (d) Far-UV CD spectra of the PCT (10 μM) in the presence and absence of NaSCN (1 mM) in the Tris-HCl buffer (5 mM).

For the case of PCT, SCN- would preferentially interact with the positively charged amine groups associated with the polyzwitterionic chains via ion pairing, as the enzyme is surrounded by these grafted polymer chains (Figure 6c). This would strengthen the hydrophobic interaction between the enzyme and the substrate. At the same time, the grafted polyzwitterionic chains can act as a protective layer to keep the conformation of enzyme stable by reducing the direct interaction between the SCNanions and the enzyme (e.g., the enzyme active site). This hypothesis is verified by the CD spectra of the PCT (Figure 6d), in which the overlap of the CD spectra in the presence and absence of SCN- suggests that the addition of SCN- has no obvious 17



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influences on the secondary structure of the PCT owing to the protection of the grafted polyzwitterionic layer. Consequently, the binding of SCN- to the grafted polyzwitterionic chains could improve the enzymatic binding affinity without obviously sacrificing the stability of the enzyme (Figures S6 and S7, Supporting Information).

Conclusion We have demonstrated that the binding affinity of the polyzwitterion-conjugated protein can be modulated by the ion-specific effects in the crowded environments through either the salting-out effects for the kosmotropic Ac- anion or the ion-pairing interactions for the chaotropic SCN- anion. In the salt-free solutions, the binding affinity of the PCT is lower than that of the BCT in the crowded environments. The kosmotropic Ac- anion is incapable of achieving a stronger binding affinity of the PCT than the BCT in the highly crowded environments, but it could reduce the difference in binding affinity between the PCT and the BCT in the highly crowded conditions compared with that in the salt-free solutions. The chaotropic SCN- anion could enhance the hydrophobic interaction between the PCT and the substrate. Meanwhile, the direct interaction between the SCN- and the enzyme could be weakened owing to the protection of the grafted polyzwitterionic layer of the PCT. This gives rise to a stronger binding affinity of the PCT than the BCT in the crowded environments.

Supporting Information: Characterizations for the synthesis of poly(carboxybetaine) 18


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conjugated α-chymotrypsin, the initial velocity with the concentration of crowder, and the measurements of enzymatic activities are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgment The financial support of the National Natural Science Foundation of China (21374110, 21574121, 21622405), the Youth Innovation Promotion Association of CAS (2013290), and the Fundamental Research Funds for the Central Universities (WK2340000066) is acknowledged.

Author Contributions †These authors contributed equally to this work.

Notes The authors declare no competing financial interests.

References and Notes 1． Myhr, K.; Ross, C.; Nyland, H.; Bendtzen, K.; Vedeler, C. Neutralizing Antibodies to Interferon (IFN) α-2a and IFN β-1a or IFN β-1b in MS are Not Cross-Reactive. Neurology 2000, 55, 1569-1572. 2. Oberg, K.; Alm, G.; Magnusson, A.; Lundqvist, G.; Theodorsson, E.; Wide, L.;

19



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

Wilander, E. Treatment of Malignant Carcinoid Tumors with Recombinant Interferon Alfa-2b: Development of Neutralizing Interferon Antibodies and Possible Loss of Antitumor Activity. J. Natl. Cancer Inst. 1989, 81, 531-535. 3. Gelfand, E. W. Antibody-Directed Therapy: Past, Present, and Future. J. Allergy Clin. Immunol. 2001, 108, S111-S116. 4. Dimitrov, D. S. In Therapeutic Proteins: Methods and Protocols; Smales, C. M., James, D. C., Eds.; Springer, 2012; Vol. 889, pp 1-26. 5. Pfister, D.; Morbidelli, M. Process for Protein PEGylation. J. Control. Release 2014, 180, 134-149. 6. Jevševar, S.; Kunstelj, M.; Porekar, V. G. PEGylation of Therapeutic Proteins. Biotechnol. J. 2010, 5, 113-128. 7. Roberts, M.; Bentley, M.; Harris, J. Chemistry for Peptide and Protein PEGylation. Adv. Drug Delivery Rev. 2012, 64, 116-127. 8. Fishburn, C. S. The Pharmacology of PEGylation: Balancing PD with PK to Generate Novel Therapeutics. J. Pharm. Sci. 2008, 97, 4167-4183. 9. Veronese, F. M. Peptide and Protein PEGylation: A Review of Problems and Solutions. Biomaterials 2001, 22, 405-417. 10. Keefe, A. J.; Jiang, S. Poly(zwitterionic)protein Conjugates Offer Increased Stability without Sacrificing Binding Affinity or Bioactivity. Nat. Chem. 2012, 4, 59-63. 20


Page 20 of 25

Page 21 of 25

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


11. Gupta, B.; Levchenko, T. S.; Torchilin, V. P. Intracellular Delivery of Large Molecules and Small Particles by Cell-Penetrating Proteins and Peptides. Adv. Drug Delivery Rev. 2005, 57, 637-651. 12. Torchilin, V. Intracellular Delivery of Protein and Peptide Therapeutics. Drug Discovery Today: Technologies 2008, 5, e95-e103. 13. Stewart, M. P.; Sharei, A.; Ding, X.; Sahay, G.; Langer, R.; Jensen, K. F. In Vitro and ex Vivo Strategies for Intracellular Delivery. Nature 2016, 538, 183-192. 14. Asaad, N.; Engberts, J. B. Cytosol-Mimetic Chemistry: Kinetics of the Trypsin-Catalyzed Hydrolysis of p-Nitrophenyl Acetate upon Addition of Polyethylene Glycol and N-tert-Butyl Acetoacetamide. J. Am. Chem. Soc. 2003, 125, 6874-6875. 15. Song, W. Q.; Liu, L. D.; Liu, G. M. Ion Specificity of Macromolecules in Crowded Environments. Soft Matter 2015, 11, 5940-5946. 16. Nakano, S.; Miyoshi, D.; Sugimoto, N. Effects of Molecular Crowding on the Structures, Interactions, and Functions of Nucleic Acids. Chem. Rev. 2013, 114, 2733-2758. 17. Lo Nostro, P.; Ninham, B. W. Hofmeister Phenomena: An Update on Ion Specificity in Biology. Chem. Rev. 2012, 112, 2286-2322. 18. Wenner, J. R.; Bloomfield, V. A. Crowding Effects on EcoRV Kinetics and Binding. Biophys. J. 1999, 77, 3234-3241.

21



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

19. Pastor, I.; Vilaseca I Font, E.; Madurga Diez, S.; Garcés, J. L.; Cascante I Serratosa, M.; Mas i Pujadas, F. Effect of Crowding by Dextrans on the Hydrolysis of N-Succinyl-l-phenyl-Ala-p-nitroanilide Catalyzed by α-Chymotrypsin. J. Phys. Chem. B 2011, 115, 1115-1121. 20. Liu, L.D.; Kou, R.; Liu, G. M. Ion Specificities of Artificial Macromolecules. Soft Matter 2017, 13, 68-80. 21 Murata, H.; Cummings, C. S.; Koepsel, R. R.; Russell, A. J. Polymer-Based Protein Engineering Can Rationally Tune Enzyme Activity, pH-Dependence, and Stability. Biomacromolecules 2013, 14, 1919-1926. 22. Zhang, Z.; Chen, S.; Jiang, S. Dual-Functional Biomimetic Materials: Nonfouling Poly(carboxybetaine) with Active Functional Groups for Protein Immobilization. Biomacromolecules 2006, 7, 3311-3315. 23. Cornish-Bowden, A., Fundamentals of Enzyme Kinetics, 3rd edition. London: Portland Press Ltd; 2004. 24. Rodríguez-Martínez, J. A.; Rivera-Rivera, I.; Solá, R. J.; Griebenow, K. Enzymatic Activity and Thermal Stability of PEG-alpha-Chymotrypsin Conjugates. Biotechnol. Lett. 2009, 31, 883-887. 25. Cummings, C. S.; Campbell, A. S.; Baker, S. L.; Carmali, S.; Murata, H,;

Russell, A. J. Design of Stomach Acid-Stable and Mucin-Binding Enzyme Polymer Conjugates. Biomacromolecules 2017, 18, 576-586.

22


Page 22 of 25

Page 23 of 25

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


26. Kostetsky, P. V. Location and Volume of the Active Site of Chymotrypsin. Biochemistry (Moscow) 2007, 72, 392-397. 27. Theillet, F. X.; Binolfi, A.; Frembgen Kesner, T.; Hingorani, K.; Sarkar, M.; Kyne, C.; Li, C.; Crowley, P. B.; Gierasch, L.; Pielak, G. J. Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs). Chem. Rev. 2014, 114, 6661-6714. 28. Parsons, D. F.; Boström, M.; Nostro, P. L.; Ninham, B. W. Hofmeister Effects: Interplay of Hydration, Nonelectrostatic Potentials, and Ion Size. Phys. Chem. Chem. Phys. 2011, 13, 12352-12367. 29. Kunz, W. Specific Ion Effects in Colloidal and Biological Systems. Curr. Opin. Colloid Interface Sci. 2010, 15, 34-39. 30. Zangi, R.; Hagen, M.; Berne, B. J. Effect of Ions on the Hydrophobic Interaction between Two Plates. J. Am. Chem. Soc. 2007, 129, 4678-4686. 31. Ui, N. Isoelectric Points and Conformation of Proteins: II. Isoelectric Focusing of α-Chymotrypsin and Its Inactive Derivative. Biochimica et Biophysica Acta (BBA)-Protein Structure 1971, 229, 582-589. 32. Collins, K. D. Ions from the Hofmeister Teries and Osmolytes: Effects on Proteins in Solution and in the Crystallization Process. Methods 2004, 34, 300-311. 33. Kou, R.; Zhang, J.; Wang, T.; Liu, G. M. Interactions between Polyelectrolyte Brushes and Hofmeister Ions: Chaotropes versus Kosmotropes. Langmuir 2015, 31,

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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

10461-10468. 34. Liu, H. L.; Liu, G. M.; Zhang, G. Z. Modulation of Self-Healing of Polyion Complex Hydrogel by Ion-Specific Effects. Chin. J. Chem. Phys. 2016, 29, 725-728. 35. Warren, J. C.; Cheatum, S. G. Effect of Neutral Salts on Enzyme Activity and Structure. Biochemistry 1966, 5, 1702-1707. 36. Bauduin, P.; Nohmie, F.; Touraud, D.; Neueder, R.; Kunz, W.; Ninham, B. W. Hofmeister Specific-Ion Effects on Enzyme Activity and Buffer pH: Horseradish Peroxidase in Citrate Buffer. J. Mol. Liq. 2006, 123, 14-19. 37. Mason, P.; Neilson, G.; Dempsey, C.; Barnes, A.; Cruickshank, J. The Hydration Structure of Guanidinium and Thiocyanate Ions: Implications for Protein Stability in Aqueous Solution. Proc. Natl. Acad. Sci. 2003, 100, 4557-4561.

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Modulation of the Binding Affinity of Polyzwitterion-Conjugated Protein

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