Effect of polymer chain density on protein-polymer conjugate

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Effect of polymer chain density on protein-polymer conjugate conformation Daniela Russo, Andrea De Angelis, Christopher J. Garvey, Frederik R. Wurm, Marie-Sousai Appavou, and Sylvain Prévost Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00184 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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Biomacromolecules

Effect of polymer chain density on proteinpolymer conjugate conformation Daniela Russo,1,2*Andrea de Angelis, 3 Christopher. J. Garvey,2 Frederick R. Wurm, 4 MarieSousai. Appavou,5, Sylvain. Prevost3 1) Consiglio Nazionale delle Ricerche & Istituto Officina dei Materiali c/o Institut Laue Langevin, 38042 Grenoble, France 2) Australian Nuclear Science and Technology Organization, New Illawarra Road, Lucas Heights NSW 2234, Australia 3) Institut Laue Langevin, 38042 Grenoble, France 4) Max-Planck-Institut für Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany 6) Jülich Centre for Neutron Science JCNS at Heinz Maier-Leibnitz Zentrum (MLZ) Forschungszentrum Jülich GmbH Lichtenbergstraße 185748 Garching, Germany

KEYWORDS:

scattering,

protein-polymer

conjugates,

structure,

conjugation,

protein

conformation, small angle scattering

Abstract Many biomedical applications employ covalent attachment to synthetic polymers to enhance the efficiency of proteins or other therapeutically active molecules. We report here the impact of polymer conjugation on the structural and thermal stability of a protein model, the

bovine

serum

albumin,

using

a

variable

number

of

linear

biodegradable

polyphosphoesters, which were covalently tethered to the protein. We observed that BSAs’ secondary structure measured by circular dichroism is independent of the conjugation. Small angle neutron scattering, however reveals a change from ellipsoid to globular shape of the

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whole complex arising from a slight compaction of the protein core and an increase of the polymer’s radius of gyration as a function of the grafting polymer density. In particular, we highlight a gradual change of the polymer conformation around the protein and elongation of the semi-major dimension of the ellipsoidal protein. Our results will contribute to the description of biophysical characteristics of a new class of biologically relevant proteinpolymer conjugates

.


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Biomacromolecules

In recent years PEGylation of proteins, i.e. covalent attachment of poly (ethylene glycol) (PEG) chains to proteins, has received increased attention.

1

After pioneering work in the

1970s2, today several PEGylated pharmaceuticals have entered the market,3,4 some of these conjugates are used for the treatment of cancer, hepatitis C and diabetes. The polymer coating on nano-carriers allows fine-tuning of interactions with various blood plasma proteins, which can be used to control non-specific cellular uptake of the nano-carriers from the bloodstream. 5 Thin films of concentrated proteins in polymer conjugates can be used as bio-sensors. Cell-free hemoglobin (Hb)-based oxygen carriers have long been proposed as blood substitutes, but their clinical use remains difficult due to problems with inefficiency and/or toxicity. Conjugation of Hb with the biocompatible polymer poly(ethylene glycol) (PEG) greatly improved their performance.6 The influence of polymer properties in conjugation with proteins can be exploited to allow more effective control of proteinpolymer properties such as as solubility, unfolding, transport and resistance to degradation. PEGylated proteins impart pharmacological advantages such as improved solubility and stability due to the polymer shield and passive targeting (enhanced permeability and retention (EPR) effect). In addition, longer circulation times and reduced administration is a benefit due to minimizing proteolytic degradation of the conjugates.3,7–9 PEGylated nanocarriers have also achieved long in vivo half-lives.10 Recently, however, PEGylation has raised several concerns.1 The occurrence of renal tubular vacuolization, caused by the accumulation of PEG in kidney cells, was reported in animal models treated with PEGylated drugs over a prolonged period of time.

11,12

This observation is attributable to PEG’s non-

biodegradability. As PEG is not biodegradable in vivo, we have developed fully degradable protein-polymer conjugates using polyphosphoesters (PPEs)13 as hydrophilic and degradable polymers14,15,16. Polyphosphoesters have been studied already in several nano-carriers and their biocompatibility was investigated by several research groups.

17–20,21Their

use in

polymer-protein conjugates had resulted in similar activities compared to PEGylated


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counterparts in a recent study. This class of polyesters degrades hydrolytically and enzymatically. PPEs22,23 as currently investigated by us, represent a promising novel and fully biodegradable alternative with a maximum of synthetic flexibility. However, more structural details on the protein denaturation or general structural behavior for PPE-protein conjugates are necessary in order to produce effective protein-based therapeutics. Despite the huge number of studied protein-polymer conjugates and the large body of literature available on their biochemical properties, very few studies have been conducted on the protein structural dynamics after conjugation to polymers.

16,24,25

However, on a more

fundamental level, it is vital to gather an understanding of the molecular level of the underlying molecular processes involved in modulating biological function in polymerconjugated proteins. On the molecular scale, the question arises as to how the polymer structurally organizes itself around the protein and how the polymer chains affect the protein 3D-structure. The currently accepted picture of PEGylated proteins invokes the concept of the shroud, in which the polymer chains are wrapped around the protein to create an effective shield that modifies properties at the interface. Although this model may be adequate for short chain lengths at higher grafting densities,2 recent works have shown that the PEGylation of a small number of chains onto the protein can lead instead to a dumbbell-type shape with the polymer chains forming random-coil structures adjacent to the protein.

26,27,28.

It is accepted

that the PEG conjugation strongly depends on the conjugate concentration29, steric clashes between PEG molecules reduce the PEG radius of gyration.

27

D. Svergun and coworkers30

show that the PEG surface acts as a shield that introduces intermolecular repulsion, which can increase with additional PEG conjugation. It has also been shown that PEGylation does not modify the change of configuration due to ligand binding and do not affect the domain structure 24,31. We have found equivalent results under the PPEgylation32. Both polymers seems to reduce the thermodynamic stability of the protein. 32,31,33,34


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Biomacromolecules

Our general aim is to contribute to the description of biophysical characteristics of a new class of biologically relevant protein-polymer conjugates and to study for the first time their interactions, low resolution structure and dynamical properties.16 We ask the general questions: how does conjugation of a synthetic polymer with variable structure and design affect the protein folding/unfolding and stability; and what are the common keys parameters that govern the stability of polymer-conjugated proteins? This paper presents the impact of the conjugation of polyphosphoesters on the structural and thermal stability of bovine serum albumin (BSA) as a model enzyme, in dilute solution where the interactions between proteins can neglected. Different equivalents of the water-soluble and linear poly (ethyl ethylene phosphate) (PEEP) were covalently attached to the lysine residues of the protein. The molecular configuration of the model-protein conjugate was measured in a diluted solution using optical spectroscopy and small angle neutron scattering. The strategic combination of these techniques provided complementary information, on the folding and the overall shape of the protein and its surrounding polymer corona respectively, which will clarify the mechanisms for the different conjugates thermal stability.

Material and methods Sample Preparation. Different polymers conjugates of Bovine Serum Albumin (BSA) were synthesized with 5, 10 and 20 attached poly (ethyl ethylene phosphate) (PEEP) polymer chains. PEEP polymer chains of 5 kDa MW are grafted on the protein surface through lysine amino acids. Details of the synthesis and characterization of PEEP and the conjugates can be found in references.14,15,22 Chains distribution is always there in a grafting-to approach and we use the mean number of polymer chains attached. We adopt the following notation for the BSA-PEEP conjugates: BSA-xp, where x is the number of polymers attached to the protein surface.


5


x

O RO P O OEt PEEP

O

O

O n

BSA borate buffer

O N

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O RO P O OEt

O

O O HN n

BSA x

BSA-PEEP conjugates

Figure 1 PEEPylation of lysine residues of proteins via NHS activated PEEP.

Tryptophan fluorescence was excited by light at a wavelength of 295 nm to maximize the quantum efficiency. Emission spectra were recorded between 280 and 550 nm at constant bandwidth (2 nm), using a Quantamaster QM4CW fluorimeter (Photon Technology International). The inner optical path of the sample was 1 cm. Measurements were performed with a wavelength increment of 0.5 nm and an integration time of 0.1 s. The protein concentration was about 0.7 µM and spectra recorded twice every 5 oC in the temperature range between 20-80 oC. All spectra were corrected for instrument response and for the Raman scattering of the solvent. Measurement with a wavelength of 280 nm was also performed and presented in the Supporting Information (S.I. Figures S4).

Circular dichroism in the far-ultraviolet range was determined with a Jasco J810. CD spectra were recorded between 190 and 260 nm for the native protein. The optical path length of the samples was 0.1 cm. The wavelength increment was 0.2 nm and a speed of 10 nm /minute. Ten measurements were performed and averaged for each sample. The protein concentration was 2.5-3.5 µM for the far UV-CD and about 0.3 µM for the near UV-CD. The temperature range for measurements was 20 oC and 80 oC. All spectra were corrected for the background, smoothed and normalized to molar ellipticity. The molar ellipticity was calculated using the expression (1), where Mw is the molecular weight, (n-1) the number of residues, C is the protein concentration (in Molar) and d is the thickness of the cell in cm. 𝜃 ∗ 𝑀𝑤

[𝜃] = (𝑛 ― 1) ∗ [𝐶] ∗ 𝑑 ∗ 100

(1)


6

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Dynamic light scattering: DLS measurements were performed with ALV CGS3 static and dynamic light scattering instrument made available within the PSCM labs at ILL. We computed the hydrodynamic radius distribution, the diffusion coefficient for all particles in solution by analyzing the experimental intensity autocorrelation function. The measurement was performed at 1 mg/ml and pH 7.5; all data were collected between 30 and 150 degrees and at 25 oC. The measurements were repeated 3 times, with run duration of 30 seconds.

Small angle neutron scattering measurements (SANS): BSA-conjugates, the BSA native protein, and the PEEP polymer were measured in a 50 mM phosphate D2O buffer at pD 7. All solutions were centrifuged at 10,000 rpm for 10 minutes before the measurements to eliminate part of aggregates. A solution at 10 mg/ml was prepared for the BSA protein, at 16 mg/ml for the PEEP (ANSTO), and at 10 mg/ml for all conjugates. For the protein concentrations and solvent conditions considered here we have assumed that there is an absence of the protein concentration-dependent effects of interparticle interactions, structure factor, and that modeling the observed scattering curve is indicative of the average shape of the protein. The BSA-20p has been also measured at a concentration of 20 mg/ml. A solution composed of a mixture of BSA and polymer with a ratio of 1:1 by mass was also measured. The corresponding BSA concentration has been determined by UV absorption spectroscopy as reported in Table S1 Supporting Information. Small angle neutron scattering data were collected on various spectrometers. The analyzed room temperature SANS data was collected on V4

35(Helmholtz

Zentrum Berlin, Germany); SANS

data from the BSA protein, the PEEP polymer and the protein/polimer mixture spectra from QUOKKA36(ANSTO, Australia); temperature dependant SANS measurements were performed on JCNS KWS237 (Heinz Maier-Leibnitz Zentrum, Munich, Germany); and SANS data with contrast variation was collected at SANS II38 (PSI, Switzerland) (section 9 Supplementary Information). The instrumental set up, and resulting q-range, for each measurement is reported in the Supplementary section, Table S2. In common, for all measurements the samples were contained in quartz cells with an inner path length of 1 mm. Sample scattering spectra were corrected for solvent or empty cell scattering and for the non uniformity of the detector response


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by normalization a flat incoherent scatterer. Finally, to correct for incoherent scattering due to the non-exchangeable hydrogen atoms (H) of the protein, a constant was subtracted from the spectra during the fitting procedure. Absolute calibration was achieved by scaling to the incident neutron flux on the sample. SANS data were first fitted using the Guinier approximation, yielding an average radius of gyration. In the following step the data were analyzed by means of the open software package SASfit39 and

SASview40 employing several models from

Pedersen41 to elliptical cores shell models and Gaussian chains, Debye for the polymer data. The detail of the fits is provided in the section 2 of the S.I. The P(r) was obtained after regularized transform of the corresponding scattering patterns using the online software package BayesApp.42


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Biomacromolecules

Results and Discussion The main aim of this work is to picture the structural features of BSA-PEEP conjugates as a function of PEEP grafting density. The structural stability of the grafting procedure is also investigated against temperature variations. To this end we performed CD and fluorescence measurements to probe secondary and tertiary conjugate structure, and SANS to characterize the protein shape/envelope and the polymer arrangement around the protein. The reductionist model of the conjugate used for modelling was an elliptical core consisting of solvent/protein surrounded by a shell consisting of solvent/PEEP. 1. Spectroscopy 1.1 Secondary structural characterization of BSA conjugates. Secondary structural characterization of BSA conjugates. To investigate the impact of the PEEPylation on the folding into secondary and tertiary structures at polymer ratios of 1:5, 1:10 and 1:20, far and near circular dichroism and fluorescence spectroscopy measurements 15 were performed at room temperature. Previous results suggested that BSA maintain its native conformation with conjugation at room temperature. New circular dichroism measurements were performed in the far (180-260 nm) and near (250 – 340 nm) UV region to investigate secondary structure respectively as a function of temperature. Figure 2 shows the temperature dependence at 222 nm of the far UV CD scan for the PEEPylated BSA compounds and the native protein in the temperature range between 25 and 80 C (the ellipticity spectra and temperature dependence of 208 nm and 108 nm are reported in the SI, Figure S3). Figure 3 shows the near UV CD scans (250-350 nm) at room temperature and 95 C. In both cases the results show that the protein secondary and local tertiary structures are not affected from the conjugation and that there is no significant difference between the various conjugates during the unfolding process. A similar result was been also observed for PEGylated BSA31 mixture solution of BSA and PEEP polymer and for a new BSA prototype conjugated (S.I., Figure S4 and Figure S5). The aging effect of the PEEP-conjugates was also investigated revealing a weak destabilization of the compound in particular for the BSA-20p (supplementary information Figure S6)


9


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Figure 2. Far UV CD molar ellipticity at 222 nm as a function of temperature for the BSA-5p, Bsa-10p and BSA-20p conjugates compared to the BSA native protein. A measurement at RT after denaturation shows the non-reversibility of the protein unfolding for all conjugates (open symbols).

a)

b)

Figure 3. Near UV-CD spectra collected at room temperature (a) and 95 C (b) for the conjugates BSA-5p, Bsa-10p and BSA-20p compared to the BSA native protein.

1.2 Tertiary structural characterization of BSA conjugates. An additional investigation of the tertiary structure was performed with standard fluorescence emission measurements (complementary data in S.I. section 4). Fluorescence signals are extremely sensitive to the microenvironment of the fluorophore, and the BSA contains two tryptophans that account for the


10

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Biomacromolecules

observed emission peak Figure 4 which shows the fluorescence emission spectra at room temperature for the BSA-5p, Bsa-10p, and BSA-20p compared to native BSA protein. Since the intensity depends on the protein concentration, all spectra were normalized to their integral in order to be compared. We first observe that PEEPylation promotes a blue shift of the maximum emission wavelength (λmax). As the amount of polymer attached to the protein increases more pronounced is the blue shift of the λmax and higher is the (normalized) fluorescence intensity, compare to native BSA. As previously speculated15 at room temperature, the blue shift is a fingerprint of a change in the environment around the fluorophores in the protein rather than an unfolding process of the tertiary structure. The increase in the intensity supports the hypothesis that the polymer arrangement around the protein plays a role on the screening of the quenching for the two tryptophans.

a)

b)


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c)

Figure 4. Fluorescence emission spectra at room temperature for all BSA-PEEPylated samples at 1 uM BSA concentration. All emission spectra have been normalized for corresponding integral. b) Fluorescence emission spectra, in the range of temperature 25 oC - 85 oC, for the

BSA-5p sample at 1 uM BSA concentration. c) λmax (nm) as a function of temperature for the BSA native protein and the 5p, 10p, 20p BSA conjugates at 0.7 uM BSA concentration.

The BSA typically shows a blue shift behavior under unfolding or changes in chemical composition of the environment. 43,44 Figure 4b show an example of fluorescence emmission spectra as a function of temperature for the BSA-5p. As already observed at room temperature the peak shifts upon heating to lower wavelengths, but this time the peak has lower emission intensity. The loss of fluorescent intensity is an effect of the temperature, while the progressive blue shift (Figure 4c) is a combination/evolution during the unfolding process where the Tryptophan (Trp) environment changes to a more hydrophobic character due to the conformational change introduced by polymer conjugation and a possibly protein aggregation following denaturation.45 The fluorescent intensity as a function of temperature confirms that the amount of the polymer has a role in the blue shift and that the conjugates in the native-like state always show a shorter λmax compare to the BSA. For temperatures above 65 oC, when the protein starts to unfold λmax tends to a plateau, which at the 75 oC reach the values of 335 nm for all conjugates. The combination of all events contributing to the fluorescence spectra including the polymer screening and the protein aggregation during the unfolding made difficult to depict a clear picture of the evolution of the tertiary structure, in particular for the conjugate BSA-20p. Therefore, in order to investigate the structural features of BSA-PEEP conjugates we performed small angle neutron scattering experiments.

2. Small Angle Scattering low resolution structural characterization


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Biomacromolecules

2.1. BSA and PEEP polymers in their monomer state. Firstly, structural characterization of both the BSA and the PEEP molecules in solution and unconjugated form has been made at room temperature and 85 oC. The high temperature allowed the characterization of the unfolded states and compares the profile with the conjugates at room temperature. In Figure 5, we show the SANS profile for the BSA at room temperature (a) and at 85°C (b) together with the scattering profiles of PEEP (c-d). All experimental curves were acquired in 100% D2O. Previous studies indicated that BSA behaves as a prolate ellipsoid with a semiminor and major axis of approximately b=20 Å and a=70 Å.46–48 In agreement with the published results, our SANS data of pure BSA at room temperature (Fig.5a) were well fitted by using a prolate ellipsoidal form factor (eq. S3 in S.I.) with b=20.40±0.01 Å and a=77.35±0.03 Å, respectively. The overall ellipticity (ν=a/b) of 4𝜋

3.800±0.002 and a molecular volume V= 3 𝜈𝑏3 of 134800±4700 Å3 could be estimated, and a Guinier analysis, according to eq.S2, give a radius of gyration equal to Rg = 32.23 ±1.2 Å and forward scattering I (0) =0.6140 +/- 0.0027 cm-1 (0.02

Effect of polymer chain density on protein-polymer conjugate

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