Investigation of the structure of protein–polymer conjugates in solution reveals the impact of protein deuteration and the size of the polymer on its thermal stability

Abstract

The conjugation of proteins with polymers offers immense biotechnological potential by creating novel macromolecules. This article presents experimental findings on the structural properties of maltose‐binding protein (MBP) conjugated with linear biodegradable polyphosphoester polymers with different molecular weights. We studied isotopic effects on both proteins and polymers. Circular dichroism and fluorescence spectroscopy and small‐angle neutron scattering reveal that the conjugation process destabilizes the protein, affecting the secondary more than the tertiary structure, even at room temperature, and that the presence of two domains in the MBP may contribute to its observed instability. Notably, unfolding temperatures differ between native MBP and the conjugates. In particular, this study sheds light on the complex interplay of factors such as the deuteration influencing protein stability and conformational changes in the conjugation processes. The perdeuteration influences the hydrogen bond network and hydrophobic interactions in the case of the MBP protein. The perdeuteration of the protein influences the hydrogen bond network and hydrophobic interactions. This is evident in the decreased thermal stability of deuterated MBP protein, in the conjugate, especially with high‐molecular‐mass polymers.

1. INTRODUCTION

In recent years, the covalent attachment of poly(ethylene glycol) (PEG) chains to proteins, known as PEGylation, has gained attention, leading to the development of various pharmaceuticals (Banerjee et al., 2012). Some PEGylated conjugates are used in diseases such as cancer, hepatitis C, and diabetes, this technique offers precise control of interactions with blood plasma proteins (Ediriweera et al., 2023; Schöttler et al., ²⁰¹⁶; Stevens et al., ²⁰²¹; Viana et al., ²⁰²³). Conjugation can affect protein stability, solubility, activity, and immunogenicity, while also altering the chemical and physical properties of the polymers. Understanding the molecular processes that modulate biological functions in these conjugates is crucial for enhanced design and broader applications. A rational approach aims to establish relations between specific chemical properties of polymers (such as molecular weight and biodegradability) and biological effects. Our prior research emphasized the important knowledge of biophysical characteristics, interactions, structure, and dynamic properties of protein–polymer conjugates (Russo et al., 2016, 2020, 2021; Russo, De Angelis, Garvey, et al., ²⁰¹⁹; Russo, De Angelis, Paciaroni, et al., ²⁰¹⁹). This understanding aimed to elucidate how polymer conjugation influences protein folding, their stability, therefore their activity. To address these concerns, we developed fully degradable protein–polymer conjugates using polyphosphoesters (PPEs) which are hydrophilic and degradable polymers (Pelosi et al., 2018; Steinbach et al., ²⁰¹⁷; Steinbach & Wurm, ²⁰¹⁶) making them a promising alternative to PEG for the development of effective protein‐based therapeutics.

Therefore, our recent studies focused on the impact of PPE, as a hydrophilic polymer, on protein folding. We conducted extensive analyses on bovine serum albumin (BSA), maltose‐binding protein (MBP), and myoglobin (Mb) to mimic the effects of water in the protein hydration shell through covalently attached polymers (Russo et al., 2016, 2020, 2021; Russo, De Angelis, Garvey, et al., ²⁰¹⁹; Russo, De Angelis, Paciaroni, et al., ²⁰¹⁹). Our findings revealed that covalently attached polymers significantly influence protein dynamics, akin to the effects of hydration water. Moreover, we observed polymer‐induced alterations in glass transition properties under specific conditions and their role in absorbing water molecules at the protein surface in the case of hydrated powder. These polymers interact with proteins at the interface, influencing protein fluctuations. The extent of this influence depends more on the rigidity of the protein than on the properties of the polymer alone.

To highlight the importance of protein size and secondary structure in conjugate stability, we investigated the structural properties of Mb, a smaller α‐helix protein (with a radius of gyration, R _g , of 16 Å) and the BSA (radius of gyration of 33 Å) (Russo et al., 2021; Russo, De Angelis, Garvey, et al., ²⁰¹⁹). Structurally, conjugation does not affect the native structure of both proteins, although we observed a decreased stability of both secondary and tertiary structures of Mb during temperature unfolding as compared to the unconjugated protein. Additionally, we detected an increased impact with increased polymer grafting density, suggesting that the size and mass of the biodegradable PPE polymer chains are of primary importance. On the other hand, the thermal unfolding pathway of the BSA protein is not affected. The higher rigidity of BSA in comparison to Mb and MBP, as demonstrated by internal dynamics experiments contributes to the observed stability of the conjugate during the unfolding process (Russo et al., 2016, 2020; Russo, De Angelis, Paciaroni, et al., ²⁰¹⁹). This intrinsic rigidity enables proteins to maintain their native conformation upon conjugation even at room temperature. We inferred that the connection between protein rigidity, secondary structure, and protein size influences the stability of protein–polymer conjugates during unfolding. Generally, a stiffer protein results in a more stable protein–polymer conjugate.

To establish general criteria, this study focuses on the structural properties of MBP conjugates. MBP is a monomeric alpha‐beta protein with 370 amino acids, featuring two distinct globular domains, and its size falls between those of Mb and BSA. Through various experiments, including circular dichroism (CD) and fluorescence measurements, we examined alterations in secondary and tertiary protein structures upon conjugation. Small‐angle neutron scattering (SANS) has also been used to elucidate the conformation and tertiary structure of the protein–polymer complex in solution. A notable advantage of our study is also the use of partially deuterated samples, enhancing the ability to explore structural aspects. This approach provides a simpler way to investigate how proteins and polymers behave together, offering potential insights to develop better protein‐based therapies in medicine (DeWitt & Maryanoff, 2017; Maria et al., ²⁰²³; Pirali et al., ²⁰¹⁹). In recent years, there has been a growing interest in the partial substitution of hydrogen by deuterium in drug development, where deuterium replaces hydrogen. It can impact the protein's structural features, including hydrogen bonds and interactions with hydrophobic regions. (Nichols et al., 2020; Ramos et al., ²⁰²¹; Tempra et al., ²⁰²³). This substitution has potential benefits, notably in improving the pharmacokinetics and reducing the toxicity of drugs, thereby minimizing adverse effects (Belete, 2022; Maria et al., ²⁰²³). The ongoing clinical investigation of deuterated compounds attests the clinical importance of deuterium substitution. Because deuterium substitution is promising in drug discovery, it is crucial to consider its effects on proteins. This modification can influence significantly the global stability and local flexibility of proteins.

2. MATERIALS AND METHODS

2.1. Sample preparation

A significant strength of neutron scattering is its ability to differentiate the scattering cross sections between hydrogen and deuterium, which enables selective deuteration to provide unique information on highly specific (hydrogenated) portions of the compound. For this purpose, about 400 mg of completely hydrogenated Maltose‐Binding Protein (MBP, MW 42500 Da) and 300 mg of 75% perdeuterated MBP (MBP‐D) were purified at the ILL Deuteration Laboratory (Grenoble, France) and used to synthesize hydrogenated and partially hydrogenated polymer–protein conjugates. Poly‐methyl‐ethylene phosphonate (PMEeP) polymers were synthesized by organocatalytic ring‐opening polymerization using the hydrogenated and deuterated monomer. REF Therefore, three different MBP‐polymer conjugates were prepared: hydrogenated MBP/hydrogenated polymer 5 and 10 kDa (MBP‐PMEeP_HH‐5 kDa; MBP‐PMEeP_HH‐10 kDa); deuterated MBP/hydrogenated polymer 5 and 10 kDa (MBP‐PMEeP_DH‐5 kDa; MBP‐PMEeP_DH‐10 kDa); and hydrogenated MBP/deuterated polymer 5 and 10 kDa (MBP‐PMEeP_HD‐5 kDa; MBP‐PMEeP_HD‐10 kDa). On average, two to three PMEeP‐chains were attached to each protein during the conjugation. Details of composition and synthesis of the samples are given in our previous work (Pelosi et al., 2018; Russo et al., ²⁰¹⁶; Steinbach & Wurm, ²⁰¹⁵). Table S1a,b summarizes the polymer and conjugate compositions. Native MBP and all conjugates were investigated in solution (10 mM phosphate buffer pH 7.5 in both H₂O and D₂O) at room temperature and as a function of temperature up to high denaturing temperature. PMEeP (i.e., the pure polymers), exhibited a molar mass of ca. 5 and 10 kDa, which was measured in its 5 kDa deuterated version as a function of temperature and used in both deuterated and hydrogenated form in the protein–polymer mixture preparation.

2.2. Optical spectroscopy techniques

CD measurements were performed in the far‐ultraviolet range using the Jasco J810 CD spectrometer (Jasco Corporation, Tokyo, Japan). CD spectra were recorded between 190 and 260 nm, from 20 to 95°C. For each sample, we used a volume of 300 μL with a 1‐mm optical path length. The wavelength increment was 0.2 nm applied with a scan rate of 10 nm/min. We performed and averaged 10 measurements of each sample at each temperature. The protein concentration was between 8 and 11 μM depending on the conjugate and we used a H₂O buffer solution. All spectra were corrected for the background, smoothed, and normalized to molar ellipticity. The molar ellipticity was calculated using expression (1), where Mw is the molecular weight, (n‐1) the number of residues (n = 370 total amino acids), C is the protein concentration (in Molar) and d is the thickness of the cell in cm.

$$\left[\theta \right]=\frac{\theta \times \mathit{Mw}}{\left(n-1\right)\times \left[C\right]\times d\times 100}$$

The cell path length was 0.1 cm.

The CD spectra were analyzed with the BestSel single spectrum analysis (https://bestsel.elte.hu). For each sample, at RT and 80°C, the percentage of alpha‐helix, beta sheets, turns, and other components was calculated. Given the composition of the sample, we assumed that the alpha‐helix percentages arise only from the protein, while the additional analyzed structure could bring a contribution due to the presence of the polymer (in particular turns and others).

Tryptophan fluorescence was excited by light at a wavelength of 295 nm to maximize the quantum efficiency and avoid the tyrosine excitation. MBP has two Trps, which both contribute to the overall fluorescence. The emission spectra were recorded between 300 and 500 nm using a Quantamaster QM4CW fluorimeter (Photon Technology International, Birmingham, USA) and an optical path of 1 cm. All measurements were performed with a wavelength increment of 0.5 nm and an integration time of 0.1 s. The protein concentration was about 3–4 μM (0.3–0.17 mg/mL). Spectra were averaged twice every 5°C in the temperature range 30–80°C. All spectra were corrected for instrument response and for the Raman scattering of the solvent.

2.3. Small‐angle neutron scattering measurements

SANS was collected from solutions of the MBP native protein, MBP‐PMEeP conjugates and a mixture of MBP and polymer (with a ratio of 1:1 by mass) in a 10 mM phosphate D₂O buffer at pD = 7. The solutions of the protein MBP and of the conjugates MBP‐PMEeP were prepared at the concentration of 5–10 mg/mL (depending on the performed experiment). All solutions were centrifuged at 10,000 rpm for 10 min before the measurements to eliminate all first small aggregates arising from the sample preparation. At room temperature, the aggregation process appearing during the measurement was estimated to affect the sample for concentrations of the order of 10%–20%. The SANS scattering profiles of the polymer PMEeP‐5 kDa as a function of temperature were published elsewhere (Russo, De Angelis, Garvey, et al., 2019).

SANS data were collected on the V4 (Keiderling & Wiedenmann, 1995) and QUOKKA (Wood et al., 2018) SANS instruments, respectively, at BERII in Berlin (dismissed) and ANSTO in Sydney Australia. Table S2 summarizes all performed experiments. The measurements performed on the QUOKKA instrument were collected using an incident neutron wavelength λ = 5 Å and at two different sample‐detector distance and acquisition times (2 m for 10 min and 14 m for 30 min). These conditions yield momentum transfers Q that cover the range 0.006–0.4 Å⁻¹. The momentum transfer is defined as Q = (4π/λ) sin (θ/2), where θ is the scattering angle. The measurements performed on the V4 instrument were collected using an incident neutron wavelength λ = 6 Å and at two different sample‐detector distance and acquisition times (3 m for 15 min and 16 m for 40 min). These conditions yield momentum transfers Q that cover the range 0.003–0.2 Å⁻¹.

Each sample was contained in fused‐silica cells with a path length of 1 mm. The isotropic 2D scattering patterns were converted to absolutely scaled scattered intensities after subtracting solvent and empty cell scattering, corrected for the non‐uniformity of the detector response by normalization to a flat incoherent scatter (poly (methyl methacrylate)), and then normalized to the incident flux. Finally, to correct for incoherent scattering due to the non‐labile hydrogen atoms in the protein, a constant was subtracted from the spectra during the different fitting procedures.

The SANS data, I(Q), in absolute scale (cm⁻¹), were analyzed by means of the open software packages SASview (Butler et al., 2012) and SASfit (Breßler et al., 2015) employing several standard models: spheres with attached Gaussian chains (Pedersen, 1997); core‐shell particles; and Debye for the polymer data. The distance distribution function P(r) was also obtained after indirect Fourier transformation of phenomenological fits to I(Q) data using the online software package BayesApp (Hansen, 2012).

3. RESULTS AND DISCUSSION

Regarding the structural properties of MBP‐PMEeP conjugates, we present here the results of CD and fluorescence measurements, which show alterations in secondary and tertiary structures of the protein, and SANS spectra that elucidate the conformation and tertiary structure of the protein–polymer complexes.

3.1. Equilibrium transition by circular dichroism and fluorescence emission

In the Supporting Information, we present molecular ellipticity data for native MBP and 5 and 10 kDa polymer conjugates at various temperatures (see Figures S1, S2, and S4–S6). These CD spectra in the peptidic region offer direct insights into the protein's secondary structure, since the measured ellipticity is not isotope‐dependent. The variation in the spectra of deuterated sample is simply attributed changes in the secondary structure. The grafted PMEeP polymer does not significantly contribute to the CD profile in the alpha‐helix wavelength range as we also demonstrate with the mixture measurements. However, it is possible that the polymer may influence the spectra in the turns and beta regions. For this reason, they will not be discussed and the inferred parameters are reported in the Supporting Information section. The spectra of the pure MBP recorded at room temperature have a typical profile of alpha‐beta proteins and are similar to those of the conjugates.

Figure 1a presents the temperature‐dependent molar ellipticity at 222 nm for the native protein, the MBP/PMEeP mixture, and all conjugates at 5 kDa. Corresponding data for the 10 kDa polymer are presented in Figure S5. At room temperature, we observe a decrease of the alpha‐helix component for all the investigated conjugates, as indicated by the intensity variation of the spectra at the 222 nm minimum. Additionally, there is a noteworthy destabilization of the deuterated MBP‐hydrogenated (DH) conjugates when compared to the fully hydrogenated conjugates. The molecular weight of the polymer amplifies the destabilization of the deuterated protein (Figure S5). This important destabilization arises from the lower stability of the deuterated protein, making it more sensitive to polymer grafting (Figure S6c and half transition temperature reported in Table S3). When comparing the conjugated molar ellipticity values at 222 nm to those of the free MBP protein or of the MBP/PMEeP mixture, at room temperature, these values correspond to temperatures in the range of 48–52°C, which marks the onset of the MBP unfolding process.

FIGURE 1.

(a) CD molar ellipticity at 222 nm is plotted as a function of temperature for the MBP protein and its conjugates with the PMEeP‐5 kDa polymer. The comparison with the MBP/PMeEP (1:1) mixture is reported. Notably, the temperature at which the unfolding starts is different for the free MBP protein and for the MBP‐conjugates. (b) Alpha helix percentage as inferred from the Bestsel fit of MBP protein, the mixture and all MBP‐conjugates at room temperature.

The temperature‐dependent changes of the molar ellipticity reveal how the MBP‐conjugation process affects the native folding of the protein, in contrast with our previous results obtained for Mb (alpha‐structure, 16 kDa) and for BSA (alpha‐structure, 66 kDa) (Russo et al., 2021; Russo, De Angelis, Paciaroni, et al., ²⁰¹⁹). The half‐transition temperatures (T _ht ), reported in Table S3 show the thermal destabilization of the secondary structure in the hydrogenated and deuterated MBP proteins and its conjugates. We first observe a difference of 3.5°C between the hydrogenated and deuterated form. While for the fully hydrogenated conjugate, we observe a value of T _m 10°C lower than for MBP, in the case of the conjugate with the deuterated MBP we probed a notable downshift of about 15°C in T _ht (with the 5 kDa polymer). Perdeuteration, the process of replacing hydrogen with deuterium in a molecule, influences the hydrogen‐bonding network and hydrophobic interactions in the case of the MBP protein. This is evident in the decreased thermal stability of the protein alone and in the conjugated form, especially with high‐molecular‐mass polymers when the deuterated protein results completely unfolded. The altered isotopic composition, where deuterium replaces hydrogen, can impact the protein's structural features, including hydrogen bonds and interactions with hydrophobic regions. Consequently, the overall stability of the protein is compromised, particularly when linked to polymers of substantial molecular mass. This observation underscores the intricate relationship between isotopic composition, molecular interactions, and thermal stability in the context of protein conjugation with high‐mass polymers.

In Figure 1a, we also highlight that the unfolding cooperativity is also modified, as indicated by the slopes of the sigmoid curves for the 5 kDa (10 kDa in Figure S5) polymer conjugates. This observation is consistent with our previous studies, where a broader transition was discussed as a characteristic feature of the coexistence of different protein conformations. This is in line with the presence of a partially unfolded protein even at room temperature.

In Figure 1b, the helix percentage for all samples at 5 kDa and at room temperature is depicted. This figure confirms a partial destabilization of the secondary structure, with a reduction of approximately 14% for the fully hydrogenated protein, and up to 25% for the deuterated protein. We remind that the fully unfolded protein (at 80°C) exhibits an estimated helix percentage of only 1.4%, suggesting that a drastic unfolding does not occur at room temperature.

Fluorescence spectroscopy gives access to the tertiary structure. An example of the Trp fluorescence spectra for MBP and MBP‐HH at 5 and 10 kDa polymer molecular weight is presented in Figure S7. As per the standard procedure, we analyzed the maximum Trp fluorescent intensity and the wavelength shift of the maximum intensity wavelength (λ_max) as a function of the temperature (Figure 2). The measured intensity decreases with temperature for both 5 kDa (Figure 2a) and 10 kDa (see Figure S8) conjugated samples. The decrease is of similar magnitude for all different conjugates for temperatures above 55°C, when the unfolding process is well established. However, at temperatures below 55°C, the emitted intensities are consistently lower for the deuterated‐MBP conjugates. This reduction in intensity is a fingerprint of a distinct Trp environment, potentially influenced by factors such as the solvent. The hydrogenated‐MBP conjugates exhibit an opposite behavior of the intensity, indicating a protected local environment that can be attributed to factors like polymer shielding or a partially unfolded polypeptide chain. In fluorescence spectroscopy, the intensity is sensitive to the micro‐environment, so a buried tryptophan is less sensitive to thermal quenching, resulting in higher emitted intensity compared to a solvent‐exposed tryptophan.

FIGURE 2.

(a) The normalized intensity at λ_max for all MBP‐PMEeP samples in function of the temperature. The Trp intensity exhibits a continuous decrease, as anticipated for solvent‐exposed Trp residues. (b) λ_max as a function of temperature for the MBP native protein and the MBP‐conjugates at 5 kDa. The onset of unfolding temperature for the MBP protein occurs at 55°C, whereas for all conjugates, it starts at 45°C.

MBP features eight Trp residues that are evenly distributed within the polypeptide chain, with five located in the alpha‐helix structure and two in the beta sheet. In the native MBP structure, most Trp residues are buried. The temperature‐dependent changes in emission wavelength at the maximum of intensity reveal interesting patterns. There is a gradual decrease within the native temperature range, followed by a sudden and distinct increase at 55°C, the previously defined transition temperature (Figure 2b). Furthermore, it is worth noting that, at 80°C, the emission wavelengths reach values lower than those of a Trp completely exposed to the solvent (i.e., ≈350 nm). Conversely, at room temperature, all conjugates exhibit a blue‐shift due to local polymer rearrangements around MBP Trp residues and their screening effect. Up to temperatures of 40–45°C, a region reminiscent of the native state is clearly defined, just before an abrupt red‐shift indicative of the unfolding process. Notably, the HD‐conjugated profile appears closer to the free MBP protein, which is also observed in the case of the 10 kDa conjugate (see Figure S8).

The CD and fluorescence results are complementary and painting a consistent picture where the MBP conjugation process destabilizes the protein, even at room temperature. This destabilization primarily affects the secondary structure, with less impact on the tertiary structure. As a result, both the MBP‐PMEeP_HH and MBP‐PMEeP_HD conjugates maintain a native‐like conformation up to 35°C before the secondary structure unfolds and up to 40–45°C before the tertiary structure undergoes changes. Consequently, the analysis of both CD and fluorescence spectra as a function of temperature suggests that the de‐structuration mechanism begins at the level of the secondary structure.

It is worth noting that deuterated MBP exhibits higher fragility during the conjugation process. The number, strength, and stability of hydrogen bonds together to the hydrophobic interaction are crucial for maintaining the stability and structure of proteins and in a completely deuterated protein those parameters could be modified under conjugation. Deuterium bonds can be slightly stronger than hydrogen bonds, but the impact of deuteration on hydrogen bond stability can depend on various factors, including the specific molecules involved, the environment, and the geometry of the hydrogen bond. Deuterium atoms are heavier than hydrogen atoms, their presence can subtly influence the overall shape and flexibility of molecules. Deuteration can alter the vibrational, rotational, and translational dynamics of molecules, which in turn affects conformational stability. Deuteration can affect the packing and orientation of molecules in hydrophobic regions.

While we lack records for the behavior of deuterated proteins in the conjugation field context, it is noteworthy that the destabilization of hydrogenated MBP was unexpected. In a previous study involving a smaller protein like Mb, we observed weakening of the conjugate, but only at temperatures beyond the native‐like temperature range. Additionally, in the case of BSA, we did not observe any impact of conjugation, even at denaturation temperatures.

MBP, as a protein, features an alpha‐helix structure with a few beta‐sheet elements and a molecular weight intermediate between Mb and BSA. It is also a two‐domain protein undergoing a ligand‐mediated conformational rearrangement, transitioning from an “open” to a “closed” structure when binding to malto‐oligosaccharides. The presence of these two domains may play a role in the observed instability, even at room temperature.

In this context, the observed unfolding scenario of the MBP‐PMEeP_DH protein is therefore defined by the correlation between the decrease in stability induced by conjugation and the alterations resulting from deuteration. This correlation sheds light on the intricate mechanisms underlying the structural dynamics of MBP‐PMEeP_DH, highlighting the interplay between various factors such as chemical modifications and isotopic substitutions.

3.2. Low‐resolution structural characterization by small‐angle scattering

It's worth noting that one of the remarkable aspects of conducting neutron scattering experiments is that selective deuteration of a macromolecule can offer unique insights into the specific hydrogenated regions of the complex. In a deuterated solvent, the fully hydrogenated samples (MBP‐PMEeP_HH) yield insights into the overall conformation of the biomacromolecule. The deuterated protein/hydrogenated polymer (MBP‐PMEeP_DH) conjugates will assist in elucidating the polymer conformation surrounding the protein surface. Meanwhile, the hydrogenated protein/deuterated polymer (MBP‐PMEeP_HD) will provide information about the conformation of the protein. Therefore, isotopic labeling combinations offer a comprehensive view of the complexes and give information about the relation between stability and deuteration.

In D₂O, the MBP protein and its conjugates aggregate with increasing temperature, therefore the data analysis will not consider the low Q range where the presence of aggregation is evident. Aggregation implies a change in the concentration, however since the real concentration is not a critic parameter in our analysis and considering that the degree of aggregation is equivalent for all the samples, we will not make other correction of assumptions on this concern. Since the optical measurements were performed in H₂O and SANS data in D₂O buffers, we will mainly compare the native and unfolded conformation.

The CD and fluorescence measurements reveal that the overall behavior of the MBP protein conjugated with 5 kDa or 10 kDa polymers are similar. For clarity, in the following paragraphs, we will primarily focus on the 5 kDa conjugates at room temperature and high temperature. Comparisons with the 10 kDa conjugates will be included in the Supporting Information section as needed. In Table 1, for clarity, we resume the important inferred structural parameters.

TABLE 1.

Inferred radius of gyration and core‐shell model structural parameter for the MBP protein, the mixture and all partially deuterated and fully hydrogenated conjugate.

	R g (RT) Guinier (Å)	R g (80°C) Debye (Å)	Core shell model
MBP	24	46
PMEeP (D)	10	10
Mixture (1:1)	25 (HD) 32 (HH)		(HH) Core = 24 Å Shell 28 Å
MBP‐PMEeP‐DH	13 18	15
MBP‐PMEeP‐HD	27	37
MBP‐PMEeP‐HH		40–46	Core = 24 Å Shell = 19 Å Shell = 22 Å (10 kDa)

3.2.1. MBP protein, PMEeP polymer, and MBP/PMEeP polymer mixture in their monomer states

In order to understand comprehensively the structure of the protein–polymer complexes, we initiated our study by measuring the free MBP protein in solution. Additionally, we examined a mixture of MBP/PMEeP (5 kDa) in a 1:1 ratio. While the behavior of the free polymer, denoted as PEEP and having a very similar chemical structure and molecular weight, has been characterized and published elsewhere (Russo, De Angelis, Garvey, et al., 2019), we now provide insights into the contribution in solution of the deuterated 5 kDa PMEeP polymer. This inclusion aids in building a complete reference for our investigation.

Figure S9a presents the temperature‐dependent scattered intensity of hydrogenated MBP measured on the QUOKKA spectrometer. Due to the presence of aggregates at intermediate temperatures, affecting the low Q region, our analysis of the scattered intensity is done only at the native temperature (27°C) and for the completely unfolded state (80–90°C). The highest and lowest temperature scattering profiles are shown in Kratky representation, which emphasizes the transition from a native globular to an unfolded conformation (Figure S9b). In the analysis of both the native and unfolded states of the scattering profiles, only data points for Q > 0.018 Å⁻¹ were considered. The radius of gyration (R _g ) of MBP at the native state, determined using the Guinier approximation, is about 24 Å (Figure S9c), consistent with values reported in the literature by Zaccai and coworkers (Zaccai et al., 2016). For the unfolded state, at 80 and 90°C, we used the Debye model, yielding R _g equal to 46 Å (Figure S9d). The details of the curve fits are provided in the Supporting Information.

The scattering profile of deuterated PMEeP polymer shows no temperature dependence within the investigated range (27–80°C, see Figure S10). This behavior is not unexpected since the deuterated polymer only includes the hydrogen atoms of CH₃ groups of the monomer and in the aromatic polymer tail. Consequently, the intensity, which depicts a Q ⁻² dependence, results in a small radius of gyration, with an approximate value of R _g  = 10 ± 1 Å, in contrast with R _g  = 32 Å observed for the fully hydrogenated polymer (Russo, De Angelis, Garvey, et al., 2019).

The analysis of the MBP/PMEeP mixture has been crucial in shedding light on the polymer's arrangement on the protein surface. As well, it provided insights into the role of covalent binding in the conjugates, a topic that we will discuss in the following sections. Two mixtures were measured, one with the hydrogenated polymer and another with the deuterated polymer, at 5 kDa. In both cases, the protein is fully hydrogenated, and the ratio is 1:1, at the concentration of 10 mg/mL. The ratio 1:1 corresponds to a ratio of 1 mole of MBP: 8.4 mole of polymers, thus a number of polymer chains per protein higher than in the investigated conjugates. Figure 3 illustrates the comparison of the MBP/PMEeP(H) and MBP/PMEeP(D) scattering profiles with that of the MBP protein. The increased intensity observed at low Q is due to aggregation. When the polymer is deuterated in the mixture, the collected intensity provides information primarily about the protein, resulting in the MBP/PMEeP(D) spectrum closely resembling that of MBP. An important finding is that the presence of free polymer in solution does not affect the protein native folding as also confirmed from the inferred R _g equal to 25 Å (Guinier approximation). On the other hand, when the polymer is hydrogenated, the intensities reflect both the protein and the polymer. A comparison of the MBP/PMEeP(H) mixture with the free polymer solution and native MBP in solution (Figure S11a) demonstrates that the polymer interacts with the protein surface, without destabilizing its globular structure, as discussed above. This interaction prevents the polymer from segregating in solution as a distinct population. Considerations regarding the preferred interaction of the polymer with the protein surface have been published in our earlier investigations and are applicable as well to MBP protein. The fit to the data with a (sphere) core‐shell model, where the radius of the protein was fixed equal to 24 Å, provides a thickness of the polymeric shell and water penetration equal to 28 Å (best fit represented in Figure S11b) which indicates a strong interaction and affinity for the protein surface.

FIGURE 3.

Comparison of the scattered intensity of the fully hydrogenated mixture with the MBP protein profile and the mixture that includes the deuterated polymer. It is evident that, in a simple mixture, the polymer has no significant impact on the globular structure of the protein.

3.2.2. MBP‐PMEeP conjugates

The samples were measured at room temperature and at different temperatures up to 80–90°C (a few examples of the scattering profiles at different temperatures are provided in part 3 of Supporting Information). To highlight specifically the influence of the polymer in both the native and denatured states of the protein, and the impact of protein deuteration, the following paragraphs present and discuss the analysis at room temperature and selected unfolding temperatures of the fully hydrogenated and partially deuterated conjugates with 5 kDa PMEeP.

For certain samples, we initially conducted an analysis at room temperature using the P(r) function, which represents the distance distribution function (Figure S12). This approach provided an initial evaluation of the size and conformational state of the conjugates.

Partially deuterated MBP‐PMEeP conjugates

In order to characterize the polymer size on the protein surface (examples of data and fits are reported in Figures S13 and S14), the conjugates with hydrogenated polymers and deuterated proteins (MBP‐PMEeP_DH) were analyzed using the mono Gaussian coil model. The Gaussian analysis was conducted at room temperature for 5 kDa (due to the absence of high‐temperature data), and at both room temperature and 80°C for the conjugates with the 10 kDa polymer. At room temperature, the PMEeP polymer surrounding the deuterated protein exhibits an R _g (radius of gyration) of 13 Å at 5 kDa. However, the analysis of the conjugates at 10 kDa reveals an increased R _g of 18 Å. This R _g decreases to a value of about 14–15 Å within the temperature range of 70–90°C, which can be associated to a thermal collapse, a phenomenon already observed for free polymers. (Russo, De Angelis, Paciaroni, et al., 2019).

It is noteworthy that, at 5 kDa, our previous observations indicated that the protein lost part of its secondary structure while retaining the tertiary structure. On the other hand, at 10 kDa, the secondary structure is completely disrupted, and the tertiary structure is significantly open. In this context, the Kratky plot representation (refer to Figure S13), demonstrates the presence of isolated chains in a Gaussian coil configuration, put in evidence by the expansion dependence on temperature and protein conformational state. At room temperature, both 5 and 10 kDa have a profile signature of a more compact arrangement compared to the 70°C. The inferred R _g values for the polymer chains, compared to the bulk polymer also reveal a strong compaction determined by both the presence of the protein chain and the grafting as constraint. The observed significant compaction can be correlated with the fact that a portion of the polymer chain is adsorbed or interacts with the protein chain while part of the polymer is like an isolated compact Gaussian coil. The grafting polymer chains onto the protein surface also affect the polymer's shape, regardless of whether the protein is in its folded or partially unfolded state. This implies that the grafting process itself introduces constraints or interactions that alter the polymer's conformation.

On the other hand, the analysis of the complementary partially deuterated complex, MBP‐PMEeP‐HD, was crucial to check the protein conformation upon conjugation, in particular at RT. Even if CD and fluorescence results pictured that the MBP keeps a native‐like conformation in the physiological temperature range, the evaluation of the size (R _g ) and of the shape is only possible through the analysis of the SANS data. At room temperature, both samples at high and low polymer molecular weight were analyzed with the Porod‐Guinier phenomenological equation suggesting a spherical‐like shape and a radius of gyration of the order of 27 Å (Figure 4). Including a small polydispersity of the size, the scattering profile, at room temperature, can also be described with a sphere of radius 29–30 Å. Both fits are good and their results report slightly higher values compared to the pure MBP. These results are consistent with the fact that the polymer is not fully deuterated and provide a small contribution to the scattering profile and with the concept of a protein maintaining a globular shape. Therefore, a core‐shell model can describe the fully hydrogenated samples. The data at high temperature (70°C), representing the unfolded MBP, are well fitted with a Gaussian chain of R _g  = 37 Å, describing a less extended chain compared to the free protein in solution. As discussed earlier for the polymer, we observed that the conformational state of the protein is also constrained by the presence of another nearby molecule. The comparison of spectra between the conjugates and the MBP solution confirms the similarity of the protein conformations at both investigated temperatures. The corresponding data are presented in Figure S15.

FIGURE 4.

Scattered intensities and best fit of the MBP‐PMEeP‐HD with 5 kDa polymer. (a) Room temperature. The straight line (red) represents the Guinier‐Porod fit and the dotted line (green) reproduces the best fit with a model of sphere. (b) Scattering profile collected at 70°C. The straight line represents the best fit with a Gaussian chain.

The analysis of the partially deuterated conjugates allows the discrimination between contributions due to the grafted polymer on the protein. This approach not only provides a reference for the analysis and understanding of the fully hydrogenated molecule but also a contribution on the deuteration impact on structure and interactions of conjugates as bio‐molecules with bio‐medical application. We are aware that the effects of an extensive deuteration on protein structure and function can be complex and may vary, depending on the protein's characteristics since it also introduces specific considerations and potential challenges, such as changes in the physical and chemical properties of molecules.

Fully hydrogenated MBP‐PMEeP conjugates

The analysis of the data of fully hydrogenated conjugates provides the structure of both protein and polymer chains. The comparison of the free protein in solution with the partially deuterated conjugates using the Kratky plot shows the effect of the polymer grafting (Figure 5 and Figure S16) at room temperature. As expected, comparing with the MBP protein profiles, we remark a broadening and asymmetry of the peak associated to a small shift of the Q _max (0.085–0.067 Å⁻¹) for the HD and HH, while the DH‐conjugate, representing the polymer, is Gaussian, as showed before. The Kratky representation, at high temperature (Figures S16), features the presence of completely unfolded chains.

FIGURE 5.

Kratky plot of the MBP protein and all the conjugates at room temperature.

In order to assess quantitatively the structural features of the entire biomolecule, we examined the scattering profile of the fully hydrogenated complex, at room temperature, using a model that characterizes the conjugate as a spherical core, representing the protein, surrounded by a concentric crown representing the polymer extension. We used the core‐shell model to evaluate the thickness and the scattering length of the shell. The fit was improved by introducing a small polydispersity of the thickness of the shell. The scattering length densities of the core (2.95 × 10⁻⁶ Å⁻², calculated using the 1ANF PDB file), and of the solvent, D₂O (6.33 × 10⁻⁶ Å⁻²) are fixed during the fitting procedure, while the thickness of the shell and the shell scattering length density (SLD) are fitted parameters within physically reasonable values. As we have determined that, at room temperature, the MBP protein maintains a native‐like structure, we applied certain constraints during the fitting procedure. Specifically, the radius of the core was initially constrained in the range of values R _c  = 25–26 Å obtained for the free MBP solution, where R _c is the radius of the core. This constraint permitted other parameters to converge and was subsequently allowed to vary in order to account for any potential minor conformational changes induced by the grafting procedure. On the other hand, we remind that MBP is a monomeric protein divided into two distinct globular domains that are connected by three short polypeptide segments and that the globular shape model is a good approximation but not a perfect one. Therefore, the inferred R _c , of the core, varies in the range of 24–25 Å, while the shell thickness, is equal to 19 Å for 5 kDa and 22 Å for 10 kDa. The thickness values are quite similar for both polymer MW, which together with the low grafting density assess that the polymer prefers to interact first with the protein surface rather than to extend toward the solvent. In line with observations in Mb‐protein systems (Russo et al., 2021) regardless of protein size, at low grafting density, a segment of the polymer chain consistently adsorbs to the protein surface due to the probability of forming hydrogen bonds around the conjugation area.

In conjunction with CD and fluorescence spectroscopy, these results confirm that the protein maintains a folded conformation similar to its native state at room temperature.

Compared to the mixture discussed in the previous paragraph, the size of the shell in the case of the conjugates is smaller, as a justification of variations in the number of polymers per protein and to the presence of grafting constraints that impose a geometrical restriction on the surface. Despite these differences, the SLD values are comparable (of the order of 5.9 × 10⁻⁶ Å⁻²) in both cases which is in accord with an effective water penetration, attributing the thickness to both water ingress and a loose polymer extension.

To estimate the number of grafted polymers (N _p ) as a function of aging time and of the radius of gyration (R _g ) of the polymers, we conducted an analysis using a model featuring a sphere with attached Gaussian chains. In agreement with the protocol of the conjugated synthesis, the N _p values, for all samples, fall in the range 2–3. The durable stability of the conjugate over time is a very important property to control, for example with storage at low temperature and in lyophilized powder form. The used polyester polymers have been ideated in order to be biodegradable, over long time, in solution. On the other hand, the R _g values of the polymer chains ranges from 15 to 18 Å with rising polymer MW and it correlates with the observed size of the chains in the DH conjugates confirming a compact two‐dimensional polymer conformation on the protein surface.

At high temperature, both the protein and the polymer exhibit a Gaussian chain configuration, as illustrated by the Kratky plot representation. In agreement, the scattering profiles, of the fully hydrogenated conjugates, in the temperature range of 70–90°C are consistent with the poly Gaussian coil model. The inferred radius of gyration falls in the range 40–46 Å, depending on the polymer molecular weight. From a comparison with the unfolded MBP (46 Å) and MBP‐PMEeP_DH (14 Å), it naturally follows that in its unfolded state the conjugated protein is more compact compared to the free protein in solution.

4. CONCLUSION

Deuterium substitution can alter the pharmacokinetics of a drug, influencing factors such as bioavailability, metabolism, and elimination. This approach has the potential to improve the efficacy and safety profiles of certain drugs. In this context, our general results contribute to the description of biophysical characteristics of biological relevant protein–polymer conjugates, encompassing the exploration of protein–polymer interactions, the role of deuteration in drug discovery, and the nuanced effects of deuteration on protein stability and flexibility. Thus, we provide advancements in both fundamental science and applied research, probing that a fully deuteration can influence significantly the global stability and local flexibility of proteins already at room temperature, but has no effect on the properties and structural arrangement of the polymer. Our results pave the basis for the design of conjugates with tailored properties, such as the need to use a co‐solvent‐free destabilized or partially unfolded protein at room temperature. We also emphasize that the molecular mass of the conjugated polymer can play an important role, as for example the ability of shifting the structure equilibrium toward a higher degree of unfolding increasing the molecular weight. In addition to this, the polymer distribution on the protein surface enhances the protein fluctuation and internal dynamics, influencing the dynamical and glass transition temperature. The combination of the polymer and deuteration impact in the protein dynamics and vibrational motion contribute to the overall stability. For this reason, in our research on protein–polymer conjugates, we emphasize the role of polymer density (varying the grafting density and the MW) on the protein surface while determining their properties. The interplay between protein size and the quantity of polymer chains significantly influences their ability to adapt to the protein surface or extend into the surrounding solvent.

We are aware that different proteins may respond differently to deuteration, some may be more sensitive than others and the structural and functional consequences need to be assessed on a case by case as today.

AUTHOR CONTRIBUTIONS

Daniela Russo: Conceptualization; investigation; writing – original draft; methodology; writing – review and editing; formal analysis; supervision; data curation; validation. Almerinda Di Venere: Formal analysis; methodology; validation. Frederik R. Wurm: Resources. Martine Moulin: Resources. Michael Härtlein: Resources. Christopher J. Garvey: Validation; methodology. José Teixeira: Methodology; validation; writing – review and editing.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing financial interest.

Supporting information

Data S1. Supporting Information.

Russo D, Di Venere A, Wurm FR, Moulin M, Härtlein M, Garvey CJ, et al. Investigation of the structure of protein–polymer conjugates in solution reveals the impact of protein deuteration and the size of the polymer on its thermal stability. Protein Science. 2024;33(6):e5032. 10.1002/pro.5032