Protein-polymer dynamics as affected by polymer coating and

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Protein-polymer dynamics as affected by polymer coating and interactions Daniela Russo, Andrea De Angelis, Alessandro Paciaroni, Bernhard Frick, Nicolas R de Souza, Frederik Wurm, and Jose Teixeira Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03636 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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Protein-polymer dynamics as affected by polymer coating and interactions D. Russoa,b*, A. De Angelisc , A. Paciaronic, B. Fricke, N. de Sousa b, F. R. Wurmd, J. Teixeiraf a) Consiglio Nazionale delle Ricerche & Istituto Officina dei Materiali c/o Institut Laue Langevin, 38042 Grenoble, France b) Australian Nuclear Science and Technology Organisation, New Illawarra Road, Lucas Heights NSW 2234, Australia c) Dipartimento di Fisica e Geologia, Università degli Studi di Perugia and CNISM, Via Pascoli, 06123 Perugia, Italy d) Max-Planck-Institut für Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany e) Institut Laue-Langevin, 38042 Grenoble, France f) Laboratoire Léon Brillouin (CEA/CNRS), CEA Saclay, 91191 Gif-sur-Yvette Cedex, France

Corresponding Author: * E-mail: [email protected]

Abstract We investigate the relaxation dynamics of protein-polymer conjugates by neutron scattering spectroscopy to understand to which extent the coating of a protein by a polymer can replace water in promoting thermal structural fluctuations. For this purpose, we compare the dynamics of protein polymer mixtures to that of conjugates with variable number of polymers covalently attached to the protein. Results show that the flexibility of the protein is larger in protein - polymer mixtures than in native protein or in conjugates, even in the dry state. Upon hydration, both the native protein and the conjugate show equivalent dynamics suggesting that the polymer grafted on the protein surface adsorbs all water molecules.

KEYWORDS. dynamic properties of protein-polymer conjugates, neutron scattering, hydration

water, protein dynamics

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Introduction Today polymers play an important role in the design of novel therapeutics. These synthetic macromolecules are coupled to drugs, which are often based on proteins, in order to optimize the therapeutic effects and to protect them in vivo against unwanted degradation and fast clearance. The typical strategy to increase the lifetime of proteins in the human body is their “PEGylation”, i.e. the covalent attachment of poly(ethylene glycol) (PEG) chains. Despite the established advantages1-5 of PEGylation, i.e. reduced immunogenicity and improved bioavailability, the biological activity of PEGylated pharmaceuticals is often dramatically decreased.4 Therefore, new strategies for protein modification were developed to increase the activity of the conjugates while keeping the other positive effects of polymer-conjugation. In this context, design of biodegradable polymer-conjugates was considered. We have recently used water-soluble and degradable poly(phosphoester)s for the preparation of protein-polymer conjugates. Such conjugates have similar enzymatic activities compared to PEGylated analogs. Moreover, they are fully biodegradable.6-7 At the fundamental level, it is important to perform a deep investigation of the biophysical characteristic of these biologically relevant protein-polymer conjugates with a special attention to their interactions, structure and internal dynamical properties. Structure and dynamics of conjugates are certainly correlated to all parameters that characterize each component, protein and polymer. A complete study must take into account size and shape of the protein, as well as size, mass, hydrophilicity and structure of the polymer chains. It is fundamental to investigate whether the polymer properties are suppressed in the conjugate, what is the role of hydration water in the dynamics of polymer-conjugated proteins, finally, if and how a full polymer coating can replace the role of water on the surface of proteins.7-8 In this paper, we study the effects of polymer conjugation on dynamical fluctuations of a protein. The complexity of the problem related to the structure and dynamics of both polymer and protein needs a plethora of techniques to achieve the whole characterization of the conjugates, ACS Paragon Plus Environment

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including the single contributions. There is a significant need to better understand how protein dynamics is impacted by different biocompatible and biodegradable polymers and to examine the general ability of polymers to mimic the effects of water in the protein hydration shell. Here, we privileged a general approach of the problem. We analyze the dynamical properties of proteins solvated with a polymer in order to better understand the relative importance of molecular mechanisms related to hydration water versus biocompatible materials. We use neutron scattering spectroscopy, namely elastic and inelastic scans to study BSA (Bovine Serum Albumin), either in its native form or conjugated with a polymer. This technique probes the emergence of relatively slow motions (1.5 ns) related to dynamic transitions, flexibility and structural relaxation of different protein-polymer complexes.7. The analysis gives a quantitative evaluation of the magnitude of mean square displacements (MSD) of hydrogen atoms, thus of the protein itself. Different conjugates were synthesized with 5, 10 and 20 attached poly(ethyl ethylene phosphate) (PEEP) polymer chains. PEEP, with molar mass 5 kDa, is grafted on the protein surface through the lysine groups via well-established NHS-chemistry (characterization details of the conjugates have been reported previously6). To compare the effect of the chemical conjugation to that of non-covalent interactions, a protein-polymer mixture in the ratio of 1:0.5 (dry weight g:g) has also been measured. All measurements were performed as a function of temperature, in the range 10-300 K, in dry powders and ≈ 50% D2O hydrated states, using the new high flux backscattering spectrometer IN16B at ILL with energy resolution of 0.9 μeV 9 (see details in SI). The main contribution to the scattered intensity of the samples is due to the large incoherent cross section of hydrogen atoms. Given the large number of hydrogen atoms in BSA, the contribution of the protein to the scattered intensity is dominant (see details in SI). We report several unexpected features of the dynamics of these biological complexes under ambient conditions.

Experimental Section Sample preparation for neutron scattering All samples were dissolved in D2O, lyophilized and kept ACS Paragon Plus Environment

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under vacuum for one day before to be stored with PO4 until measurements. Before the measurement, the dry samples were sealed in a standard flat aluminum holder 0.2 mm thickness. To hydrate the samples, we have exposed them to an atmosphere of 100% humidity, for a long period of time. As a function of exposed time, we could determine the various hydration levels, from the weight difference between the same sample before (dry) and after the exposition (hydrated). To prepare the Mixture matrix we dissolved the protein and the polymer, in the desired quantity, in D2O then we proceed to lyophilization. The obtained powder was kept under secondary vacuum for one day and stored with PO4 until measurement. The ratio BSA: PEEP in the mixture is about 1:10.

Elastic and quasi-elastic neutron scattering experiment Neutron scattering experiments were performed on the new high flux backscattering spectrometer IN16B4 at ILL with energy resolution of 0.9 micro eV. Elastic and Inelastic fix window scans were recorded in a range of temperature between 50 and 300 K and a heating rate of 1.5 K/min. The collected signal was measured over a wave vector Q range extending from 0.1 to 1.8 Å-1 where Q= 4πsinθ/λ is the elastic momentum exchange, 2θ is the scattering angle and λ is the wavelength of the incident neutrons. All spectra were corrected for detector efficiency and normalized using the respective measurement at 50 K. The resulting data were corrected using ILL LAMP programs5 and analyzed. The data collected on EMU and BASIS Backscattering were recorded in the Q range 0.2-2 Å-1, and between 50 and 300 K, with an energy resolution of 3.5 ueV

Results and Discussion In order to characterize the perturbation of protein-polymer dynamics due to the presence of the polymer at different concentrations, we measured the scattering of completely hydrogenated complexes by Elastic Incoherent Neutron scattering [7]. Here, Elastic scattering is defined as the intensity that


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arises from atoms that are immobile on the timescale defined by the instrumental resolution. Evolution of the Elastic Intensity versus temperature is related to active movements occurring in different conformations of protein conjugates, which arise from perturbation of the native environment. Figure 1a depicts the measured elastic intensity integrated over all scattering angles of BSA native protein, PEEP polymer and the mixture BSA-polymer, all in dry state. It provides important information on the nature of the dynamic interactions between protein and polymer. The decrease from unity of the integrated elastic intensity with increasing temperature is directly related to the mobility of the system10. It is clear, from the figure, that mobility increases when going from the protein, to the polymer and then to the mixture. BSA elastic scattering intensity decreases smoothly while there are larger discontinuities of slope for both polymer and mixture. These changes are observed at 100 K for BSA and at 150 K for both PEEP and the BSA-polymer mixture, i.e. in the typical temperature domain where the dynamics of the methyl groups present in all the samples become visible. An additional change of slope appears in the intensities of both polymer and polymer-protein mixture at 230 K, just in correspondence with the polymer glass transition temperature Tg. This coincidence indicates that polymer dynamics plays a central role also in the mixture.

The trend emerging from the simple integrated elastic intensity is very useful, as it provides model-independent information. However, to quantify the amplitude of structural fluctuations one has to resort to some approximation to interpret the Q-dependence of the elastic intensity and obtain the mean square displacements of hydrogen atoms, = MSD. This is done, by applying the Gaussian approximation, as illustrated in Figure 1b, where we compare values of dry BSA native protein, dry PEEP polymer and dry BSA-polymer mixture. Figure 1b confirms that the amplitude of structural fluctuations in the mixture is larger than that of the constituents alone, denoting some form of interaction between protein and polymer. Indeed, in the absence of interaction, we expect the extent of the mixture dynamics to be intermediate between those of protein and polymer. ACS Paragon Plus Environment

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Even though we cannot discriminate between the mean squared displacements of each component of the mixture i.e. native BSA and PEEP, since both contribute to the signal, this trend indicates that the interaction between the two components has the net effect of enhancing the total dynamics of the system. Owing to the high intrinsic mobility of the polymer compared to that of the folded protein, it is likely that the former reinforces or enhances indirectly the dynamics of the latter, mainly through the hydrogen bonds formed between the phosphate groups of the polymer chain and the protein polar groups. In this picture, the interaction between the aromatic phenyl group at the end of the polymer and the protein side-chains can be safely neglected, due to the large number of hydrophilic phosphate groups of the PEEP chains. At this stage, we believe that the presence of BSA can also affect the polymer dynamics as is larger than both and . As we will discuss later, the protein decouples the motion of neighboring PEEP chains close to its surface, thus promoting the global dynamics. Comparing more in detail MSD of dry and 20% D2O hydrated PEEP, we observe a clear enhancement of the polymer flexibility at 150 K (see Figure S1 in SI) with amplitudes comparable to those of the dry mixture up to 250 K. We do not exclude the possibility that, because of its high hydrophilicity, PEEP can sequester a certain amount of water molecules from the protein surface (structural water and strongly attached water), thus contributing to the observed dynamics. The resultant picture is that presence of BSA protein enhances polymer dynamics (master role) and, at the same time, BSA atoms with a “frozen” dynamic in the observed time scale, experience a whole “melt” due to the interaction of the polymer with the protein on its surface (slave role). The enhancement of dynamics measured in the mixture above 230 K is very peculiar when compared to what is observed in the hydrated protein. In the latter case, BSA fluctuations are promoted whereas the degrees of freedom of the solvent are constrained with respect to the bulk

11-14.

This picture is confirmed by the results

displayed in Figure 2, where the mean square displacement of the dry polymer/BSA mixture is compared to that of 50% D2O hydrated BSA. The result is unexpected: the dynamics of both hydrated ACS Paragon Plus Environment

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BSA and the mixture are identical until 230 K, i.e. the polymer Tg , while above this temperature the mixture exhibits definitely larger amplitudes. This behavior can arise either from a direct contribution of the polymer dynamics above Tg or from a different relative level of hydration, i.e. it originates from the interaction with the polymer in one case and with water molecules in the other. A combination of both effects is also possible. We conclude that both hydration and inclusion in a polymer matrix affect similarly the dynamics of the protein. Complementary measurements performed on EMU backscattering spectrometer confirmed our previous results (see details in SI, figure S2).

a)

b) Figure 1. a) Temperature dependence of Elastic scattered intensity, I(T), of dry samples, integrated over Q. b) Mean square displacement, , of hydrogen atoms obtained from I(T) assuming Gaussian approximation I(T)= exp (-Q2). Discontinuities of slopes reveal dynamic transitions, thus dynamic activation of BSA, PEEP and their mixture. Glass transition of PEEP is seen at 230 K.


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a)

b) Figure 2. Comparison between hydrated BSA and dry poly-BSA mixture. Same quantities as in figure 1 for hydrated BSA and dry BSA-PEEP mixture. Due to the presence of PEEP, at high temperature, dynamics of the dry mixture is larger than that of hydrated BSA. The dynamic enhancement takes place at the glass transition temperature of the polymer.

In order to clarify the interaction between polymer and protein, particularly on the surface of the protein, which is at the origin of the enhancement of fluctuations, we compared the protein-polymer mixture dynamics to that of protein-polymer conjugates, where the polymer is chemically grafted by covalent bonds to specific sites on the surface of BSA. The surface of BSA protein estimated by the Area Solvent Accessible methods is around 1400 Å2 , which corresponds to accessible areas of 280, 140 or 70 Å2 per polymer chain, as a function of the number of grafted chains, supposed uniformly distributed on the surface. The resultant relatively large distances between grafted chains explains the observed enhanced dynamics. Indeed, as shown in figure 3, mobility of conjugates increases with the ACS Paragon Plus Environment

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number of polymer chains grafted on the surface of the protein, as if the protein dynamics (dominant scattering contribution) is gradually enhanced by the addition of polymer chains forming hydrogen bonds with BSA side-chains. The same behavior has been observed, in the ps time scale, by quasielastic neutron scattering experiments (SI, figure S6). It is worth noting that, within the nanosecond timescale, MSD in dry mixtures is always larger than in dry conjugates and that dry BSA shows the lowest MSD.


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b

c) Figure 3. Effect of conjugation on dynamics of dry samples. a) and b) Same quantities as in preceding figures for BSA grafted with different number (5 to 20) of polymer chains compared to dry BSA and dry mixtures (as shown above) Dynamic enhancement increases with the number of grafted chains. However, the faster dynamics is always observed in dry mixtures of BSA and polymer; c) Temperature dependence of Inelastic scattered intensity for all dry samples, integrated over Q, for Δω = 2 μeV.

The observed enhancement of the flexibility of the conjugates can be reasonably explained if we describe the structure of the protein-polymer conjugates in terms of a core shell model, with the thickness of the polymer shell around the protein increasing as a function of the number of grafted polymer chains (preliminary data from SANS, Russo et al). When the number of polymer chains increases, there is less room for them to interact with the protein surface due to steric hindrance (and to minimization of interpolymer interaction due to repulsive electrostatic energy) so that they can “freely” explore conformations in the free space between conjugated proteins. In fact, as we expect the MSD of ACS Paragon Plus Environment

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protein-polymer conjugates tends to the dynamics of the simple dry polymer as the number of conjugated polymers increases (See figure S3 in the SI) However, the temperature dependence of the scattered intensity confirms that the whole conjugate is more flexible, given the protein contribution. On another hand, the covalent bond between polymer chains and protein surface significantly reduces the degrees of freedom of the hydrogen bond network formed between PO4 groups and protein sidechains. . Necessarily, the arrangement of the polymer chain on the protein surface is much different than in the case of the protein-polymer mixture, since now part of the chain is grafted to the surface, thus making the conjugates MSD smaller than that of the mixture for a number of polymer chains smaller than 20. The behavior of the integrated intensity (Figure 3a) confirms that the total flexibilities of the protein-polymer mixture and BSA-20p are comparable In agreement with the results of the previous paragraph, we speculate that, for protein dynamics, addition of polymer chains is similar to an increase of hydration water 15. However, given the differences between the mixture and the 10-polymer conjugated BSA, local interactions and global distributions of the polymer on the protein surface play also a crucial role. Additional measurements were performed on BASIS backscattering instrument and confirmed our previous results (see details in SI, figure S4). Figure 3c shows the integrated inelastic intensity measured at energy Δω = 2 μeV. 9The measurement was performed simultaneously with the elastic scan. In agreement with the observed elastic behavior, the inelastic intensity monotonically increases with temperature for dry BSA conjugates and for dry and hydrated polymers. A clear relaxation is evident at about 180 K and 150 K for the mixture and for the polymer respectively. Since we observed that polymer grafting enhances BSA fluctuations, in order to understand whether addition of water molecules could still have a role in the protein dynamics, we measured the same protein/polymer mixture at two different hydration levels: 55% and 100% (Figure 4a). Effects of hydration in the mixture are peculiar. We do not observe any drastic enhancement of the fluctuation at 50% hydration level, rather a decrease of amplitude for temperatures above 250K. As previously ACS Paragon Plus Environment

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speculated, polymer fluctuations enhance the dynamics of the BSA protein. Being the protein surface coated by the polymer, we expect that the water molecules added to the mixture will mainly be absorbed by the exposed hydrophilic sites of the polymer (mainly phosphate groups), rather than interacting with the protein side-chains, due to the quite large hydrophilic character of the former.16 Therefore, given the hydration temperature dependence, it is likely that added water molecules will form a cluster around or react with the polymer hydrophilic groups, eventually modifying the glass transition temperature of the system. This rather coordinated water molecules would then make the shell of polymer chains around the protein surface even less mobile than in the case of the dry proteinpolymer mixture. Figure 4b compares BSA, conjugated and mixture hydrated at 55 % (data of 100% hydrated mixture are depicted in SI, figure S5). The MSD profile shows that all samples behave similarly, confirming the hypothesis that water remains absorbed only on the polymer interface. The inelastic intensity (Figure 4c) shows a relaxation dynamics occurring at 220 K, as expected from hydrated macromolecules. 12,15

a

b


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c) Figure 4. Effect of hydration on dynamics a) and b) Mean square displacement, , of hydrogen atoms comparing dry and hydrated mixtures BSA/polymer to hydrated samples of BSA, PEEP and grafted BSA. The faster dynamics is observed in dry mixture ; c) Temperature dependence of Inelastic scattered intensity, for the hydrated samples, integrated over Q, for Δω = 2 μeV.

Conclusion. Protein-polymer conjugates combine the biological function of proteins with the more versatile handling of synthetic polymers. Insights into the dynamics and structure of proteins coated with polymers contribute to a better knowledge of the role of hydration in protein stability and activity. As well, they lead to important applications and development of bio-therapeutic and food science technology. Nano-systems formed by polymer-protein complexes are particularly suited for drug delivery and pharmaceutical applications. The interest of using PEEP polymer is due to its biodegradability and biocompatibility. In this work, we provide evidence that protein flexibility, in terms of mean square fluctuations, increases with the number of attached polymer chains. We show that interactions between protein and polymers play an important role, enhancing the magnitude of protein dynamics. We highlight that polymer coating has the same effect on the protein dynamics than hydration water. Conjugates, upon hydration, show an equivalent relaxation, suggesting that the polymer at the protein surface adsorbs all water molecules. We think that the increase of the protein dynamics is a general finding. It is possible that different polymer varieties (linear, branched, charged) can affect dynamics in a different manner,


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although always enhancing global dynamics.

ASSOCIATED CONTENT Supplementary material (sample preparation, neutron scattering experiment, data analysis) is available. ACKNOWLEDGMENT D.R. thanks SNS for beam time allocation on BASIS, Dr. Nina Jalarvo and Dr. Eugene Mamontov for performing the preliminary measurements on BASIS. D.R. thanks the Australian Centre for Neutron Scattering and Institut Laue-Langevin for beam time allocation. A.de A. is grateful to Institut Laue-Langevin for the financial support during his permanence at ILL to finish his Master thesis in Physics. F.R.W. thanks T Steinbach for help and assistance in BSA conjugates sample preparations. .


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TOC

Schematic representation of linear polymer conjugation


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Protein-polymer dynamics as affected by polymer coating and

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