Investigating the viscoelastic properties of aqueous protein systems by Brillouin spectroscopy and molecular dynamics

Hanlon, Dillon (2023) Investigating the viscoelastic properties of aqueous protein systems by Brillouin spectroscopy and molecular dynamics. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

In this study, we explore how proteins and water molecules interact which could be of importance in fully understanding biological processes. By using Brillouin light scattering and molecular dynamics together, we aim to learn more about these interactions. Specifically, Brillouin spectroscopy was employed to investigate the temperature dependence of the viscoelastic properties of gastropod mucus, in both its natural and hydrated and dehydrated states, and in a bacterial cell lysate solution. Additionally, time-dependent Brillouin scattering studies were conducted on dehydrated mucus. The concentration of proteins in both the hydrated and dehydrated states of gastropod mucus was determined using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy. Molecular dynamics (MD) simulations were also performed on protein-water systems to explore bulk viscoelastic properties similar to those obtained from Brillouin spectroscopy. The Brillouin spectra of natural gastropod snail mucus revealed two peaks. The first peak, located near 8.0 GHz, and was attributed to the longitudinal acoustic mode of the liquid mucus and persisted throughout the temperature range. The second peak, observed only at temperatures T ≤ −2.5`C and having a shift of approximately 18.0 GHz, indicated the presence of a phase transition. At this temperature, anomalies in the temperature dependent parameters of the longitudinal acoustic mode and the associated viscoelastic properties, as well as the emergence of the ice peak, suggested a transition from a viscous liquid state to a coexistence of liquid mucus and solid ice phases. The incomplete phase transition, as indicated by the presence of an ice peak at -2.5`C, was attributed to glycoprotein-water interactions. Moreover, the Brillouin scattering results indicated that water molecules bind to glycoproteins in the mucus at temperatures above the freezing point, leading to a reduced capacity of bound water to facilitate freezing. Consequently, a gradual liquid-solid transition and depression of the freezing point occurred. Temperature dependent Brillouin scattering on diluted and dehydrated mucus provide complimentary results to that of the natural mucus, while also adding results for the concentration dependence of snail mucus as a function of time. As the dilutions increased, the spectral parameters, frequency shift and full width at half maximum (FWHM), both decreased. Furthermore, with the addition of more water to the system, the freezing point depression observed in the natural snail mucus, increased from -2.5`C up to -1.0`C. The ice peak remained unchanged with varying dilution. Dehydrated mucus displayed a single peak attributed to the longitudinal acoustic mode of liquid mucus over the entirety of the experiment. The frequency shift increased as the protein concentration increased, as indicated by ATR data. Likewise, the FWHM also increased as protein concentration increased. Results from the dehydration experiments are indicative of a transition to a gel like phase once a protein concentration of ⇥ 50 wt% glycoproteins was reached. Furthermore, bacterial (E. coli) cell lysate in solution with water was investigated using Brillouin spectroscopy as a function of temperature. A single peak corresponding to the longitudinal acoustic mode of the fluid was observed, with frequency shifts consistently smaller than those of water throughout the temperature range studied. In addition to experimental studies, molecular dynamic simulations were employed to explore the viscoelastic properties of protein-water systems. The simulations covered a temperature range of 280K to 340K and revealed strong temperature dependence of properties such as bulk modulus, speed of sound, and viscosity. We observed a consistent increase in the bulk modulus, speed of sound, and viscosity as we increased the protein concentration. Notably, our molecular dynamics simulation results closely resemble the trends and behaviour observed in Brillouin scattering experiments conducted on aqueous protein solutions. This similarity in MD and experimental work validates the utility of simulations in exploring the viscoelastic properties of protein water solutions. Consequently, our work provides a strong rationale for using computer simulations with experimental techniques, o↵ering potential for advancing our understanding of both simple and complex systems. Collectively, this comprehensive study sheds light on the viscoelastic properties of di↵erent biological systems, providing valuable insights into phase transitions, water interactions, and the influence of protein concentration on their mechanical behavior.

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