Unlocking complexity through neutron scattering: Structure and dynamics of protein-polymer conjugates

Abstract

Protein-polymer conjugates are engineered to enhance the pharmacokinetics and stability of biopharmaceuticals. This review delves into the structural and dynamic characteristics of protein-polymer conjugates, employing advanced techniques such as neutron scattering, circular dichroism, and fluorescence spectroscopy. Here, we focus on model proteins like maltose-binding protein (MBP), bovine serum albumin (BSA), and myoglobin (Mb) conjugated with hydrolysable polyphosphoesters (PPEs) to generate fully degradable polymer-protein conjugates. The study underscores the influence of factors such as the polymers' molar mass, grafting density, hydration levels, and deuteration on protein stability, flexibility, and thermal properties. The study demonstrates that the degree of polymerization, solvation properties, and isotopic composition play crucial roles in determining the behavior of protein-polymer complexes. In particular, neutron scattering techniques are invaluable in unraveling the interaction mechanisms, providing valuable insights that can inform the optimization of protein-polymer conjugates for therapeutic applications.

Keywords: dynamical fluctuation; isotopic deuteration; neutron scattering; protein–polymer conjugates; radius of gyration.

FIGURE 1

The chemical structures of poyl(ethylene glycol) and PPEs (R typically CH3, CH2CH3, or OCH2CH3).

FIGURE 2

(a) Bovine serum albumin, BSA 583 amino acids; (b) maltose‐binding protein, MBP 392 amino acids; (c) myoglobin, Mb 152 amino acids.

FIGURE 3

The CD molar ellipticity at 222 nm, a marker of α‐helical content, is plotted as a function of temperature for (a) fully hydrogenated and completely deuterated MBP protein, and (b) partially deuterated MBP conjugates with 5 and 10 kDa polymers. The fully deuterated MBP protein shows a slight destabilization of α‐helical structure, which becomes more pronounced in the polymer conjugates, reflecting the impact of both conjugation and deuteration on protein stability (Russo et al. 2024). Reprinted from Haertlein et al. (2016).

FIGURE 4

The CD molar ellipticity at 222 nm, indicative of α‐helical content, is plotted as a function of temperature for MBP, BSA, myoglobin (Mb), and their conjugates with the PMEeP‐5kDa polymer. (a, b) MBP, fully hydrogenated and partially deuterated (polymer) conjugates show significant secondary structural changes at room temperature, indicating a loss of α‐helicity upon polymer conjugation. This suggests structural destabilization even under mild conditions. (c, d) Myoglobin and BSA conjugates demonstrate higher thermal stability compared to the native proteins, with the polymer contributing to resistance against unfolding and preserving α‐helical structure at elevated temperatures (Russo et al. ; Russo et al. ; Russo et al. 2024). Reprinted with permission from Russo et al. (2024).

FIGURE 5

The ratio D _max/R _g , derived from the P(r) analysis, is used to define the ellipticity of the particles. The ellipticity is shown for (a) BSA (Reprinted with permission from Russo et al. (2024)) and (b) myoglobin, along with their respective conjugates, as a function of grafted polymer size and type. This highlights the influence of polymer conjugation on the shape and structural compactness of the protein particles (Russo et al. ; Russo et al. 2024). Reprinted from Russo et al. (2019a).

FIGURE 6

(a) Schematic core‐shell representation, where the protein forms the core and the surrounding polymer, along with associated water molecules, constitutes the shell. (b, c) Small‐angle neutron scattering results fitted using the core‐shell model: (b) core radius, representing the protein size, and (c) shell thickness, representing the polymer layer and associated water molecules. Data correspond to myoglobin conjugates (Russo et al. 2021). Reprinted from Russo et al. (2019a).

FIGURE 7

Radius of gyration (R _g ) of the polymer molecules grafted to the protein, as determined from the Pedersen model for a sphere with attached Gaussian chains. (a) Data for myoglobin conjugates (Reprinted from Russo et al. (2019a)). (b) Data for BSA conjugates (Reprinted with permission from Russo et al. (2024)). For reference, the radius of gyration of the bulk polymer is approximately 32.0 ± 0.3 Å.

FIGURE 8

Scattered intensity profiles are compared for the hydrogenated MBP protein, the fully hydrogenated MBP–protein mixture, and the mixture containing the deuterated polymer (PMEEP(D)). Reprinted from Pirali et al. (2019). The analysis shows that the polymer has no significant effect on the globular structure of the MBP protein in the mixtures (Russo et al. 2024).

FIGURE 9

Dry state: the effect of conjugation on the dynamics. The elastic intensity probed in an elastic neutron scattering experiment arises from atoms, which are immobile on the timescale of the instrumental resolution. Elastic scans give access to the transition temperatures and mean square displacements. The figures report integrated elastic intensity as a function of temperature for dry conjugates and their components for MBP, BSA, and Mb protein. (a) MBP conjugates have about four grafted polymers. The polymer glass transition can be observed at around 240 K. A smooth and linear decrease of the intensity as a function of temperature is characteristic for the native hydrogenated MBP protein, the MBP(H)‐PMeEP(H)_5kDa (fully hydrogenated sample, polymer MW = 5 kDa), the MBP(H)‐PMeEP(H)_10kDa (fully hydrogenated sample, polymer MW = 10 kDa), and the MBP(H)‐PMeEP(D)_10kDa (partially deuterated sample, enhancing the protein dynamics). We observe a lack of abrupt dynamical transition and that increasing the polymer molecular weight decreases the elastic intensity as a signature of increased flexibility and fast dynamics (Russo et al. 2016). Reprinted with permission from Russo et al. (2019b). (b) BSA grafted with different numbers (5–20) of polymer chains (5 kDa) compared to those in dry BSA and dry mixtures. Dynamic enhancement increases with the number of grafted chains and in the mixture (Russo et al. 2019b). Reprinted with permission from Henzler‐Wildman and Kern (2007). (c) Mb grafted with different numbers (5–20) of polymer chains (5 kDa) compared to those in dry Mb. In accordance with the previous figure, we observe a decrease in the elastic intensity. Compared to the BSA, we remark a different temperature dependence, most likely related to a distinct polymer conformation on the protein surface (Russo et al. 2020). Reprinted from Russo et al. (2016).

FIGURE 10

Hydrated state: the effect of conjugation on the dynamics Integrated elastic intensity as a function of temperature for hydrated conjugates compared to dry and hydrated native MBP. Reprinted with permission from Russo et al. (2019b). All hydrated curves show an abrupt change in the elastic intensity as a fingerprint of dynamical transition. That is, the larger the loss of elastic intensity the higher is the flexibility of the system. The dynamical transition of hydrated MBP is observed at T = 200 K while for all other samples, given the presence of the polymer, it is observed at T > 225 K.