(B-56) Biophysical characterization of polymer-protein interactions through small-angle x-ray scattering and quartz crystal microbalance

Introduction:: Proteins’ precise tertiary structure enable versatile functionality; however, proteins are unstable and prone to denaturation outside their native conditions. There is a significant interest in the development of polymers capable of preferentially interacting with proteins to maintain their native structure in stressed conditions, such as elevated temperature. Here, we combine two techniques, quartz crystal microbalance with dissipation (QCMD) and small-angle x-ray scattering (SAXS) to study the biophysical interactions between polymers identified in a previous study and the enzyme horseradish peroxidase (HRP).^1,2

Materials and Methods:: Polymers: Monomers included methyl methacrylate (MMA), poly(ethylene glycol) (n) monomethyl ether monomethacrylate (PEGMA), [2-(methacryloyloxy)ethyl] tri-methylammonium chloride solution (TMAEMA), and 3-sulfopropyl meth-acrylate potassium (SPMA). Copolymers G3P1 (feed ratio: 59 mol% MMA, 12 mol% PEGMA, 29 mol% TMAEMA; degree of polymerization (DP) = 75) and G4P2 (feed ratio: 33 mol% MMA, 39 mol% TMAEMA, 28 mol% SPMA; DP = 150) were synthesized via automated photoinduced electron-transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization as previously described. ²

QCMD/SAXS: HRP was immobilized on gold sensors on a Q-Sense Omega QCMD following the procedure of Su et al.³ Flow rate was 10 μl/min, layer mass was calculated via the Sauerbrey equation, and the Voigt model was used to evaluate shear modulus of the adsorbed layer. SAXS experiments were conducted at the National Synchrotron Light Source II of Brookhaven National Laboratory (Upton, NY). HRP concentration was fixed at 1 mg/ml in 50mM sodium acetate buffer. HRP + copolymer samples were codissolved to achieve 1:1, 5:1, 10:1, and 50:1 HRP: copolymer ratios. SAXS used 15.14 keV x-rays (λ=0.8189 Å) and three Pilatus 1 M detectors for data collection. Thermal stability was tested by heating samples at 65°C for 1 h in a hot water bath.


Results, Conclusions, and Discussions:: Results and Discussion: QCMD shows an irreversible increase in adsorbed mass when G3P1 is flowed over deposited HRP, providing evidence of polymer-protein complexation. Reversing the deposition order reduced adsorbed mass, indicating HRP conformation governs polymer-protein interactions. Heated HRP adsorbed significantly more copolymer mass than native HRP, which we propose is due to HRP partially unfolding upon heating and hybridizing with G3P1 while refolding upon cooling. SAXS normalized Kratky and Porod plots demonstrated that HRP – G3P1 mixtures post-heat exposure form globular structures that closely resemble unheated native HRP. HRP-G4P2 mixtures with and without heating showed no evidence of HRP stabilization.

Conclusions: QCMD and SAXS were used to gather complementary information on structure, flexibility, and interactions between HRP and copolymers. Copolymer G3P1 appears to form complexes with HRP when the codissolved molecules are heated and cooled, resulting in polymer-protein complexes with similar hydrodynamic radii and flexibility to unheated native HRP, as evidenced by SAXS. QCMD confirmed that these complexes yield significantly increased thickness and differences in adsorption and dissipation kinetics compared to native HRP.

Acknowledgements (Optional): : Acknowledgements: NSF CBET award #2009942 and NSF DMREF award #2118860

References (Optional): : [1] Upadhya R, et al. J Biomed Mater Res A 2023;111,4;440-450 [2] Tamasi MJ, et al. Adv Mater. 2022;34:2201809. [3] Su X, Zong Y, Richter R, Knoll W. J Colloid Interface Sci. 2005;287(1):35-42.