Molecular crowding facilitates assembly of spidroin-like proteins through phase separation

Gaining insights into the processes that transform dispersed biopolymers into well-ordered structures, such as soluble spidroin-proteins to spider silk threads, is essential for attempts to understand their biological function and to mimic their unique properties. One of these processes is liquid-liquid phase separation, which can act as an intermediate step for molecular assembly. We have shown that a self-coacervation step that occurs at a very high protein concentration (> 200 gl(-1)) is crucial for the fiber assembly of an engineered triblock silk-like molecule. In this study, we demonstrate that the addition of a crowding agent lowers the concentration at which coacervation occurs by almost two orders of magnitude. Coacervates induced by addition of a crowding agent are functional in terms of fiber formation, and the crowding agent appears to affect the process solely by increasing the effective concentration of the protein. Furthermore, induction at lower concentrations allows us to study the thermodynamics of the system, which provides insights into the coacervation mechanism. We suggest that this approach will be valuable for studies of biological coacervating systems in general.Peer reviewe

pH-Induced Changes in Polypeptide Conformation : Force-Field Comparison with Experimental Validation

Microsecond-long all-atom molecular dynamics (MD) simulations, circular dichroism, laser Doppler velocimetry, and dynamic light-scattering techniques have been used to investigate pH-induced changes in the secondary structure, charge, and conformation of poly l-lysine (PLL) and poly l-glutamic acid (PGA). The employed combination of the experimental methods reveals for both PLL and PGA a narrow pH range at which they are charged enough to form stable colloidal suspensions, maintaining their α-helix content above 60%; an elevated charge state of the peptides required for colloidal stability promotes the peptide solvation as a random coil. To obtain a more microscopic view on the conformations and to verify the modeling performance, peptide secondary structure and conformations rising in MD simulations are also examined using three different force fields, i.e., OPLS-AA, CHARMM27, and AMBER99SB-ILDNP. Ramachandran plots reveal that in the examined setup the α-helix content is systematically overestimated in CHARMM27, while OPLS-AA overestimates the β-sheet fraction at lower ionization degrees. At high ionization degrees, the OPLS-AA force-field-predicted secondary structure fractions match the experimentally measured distribution most closely. However, the pH-induced changes in PLL and PGA secondary structure are reasonably captured only by the AMBER99SB-ILDNP force field, with the exception of the fully charged PGA in which the α-helix content is overestimated. The comparison to simulations results shows that the examined force fields involve significant deviations in their predictions for charged homopolypeptides. The detailed mapping of secondary structure dependency on pH for the polypeptides, especially finding the stable colloidal α-helical regime for both examined peptides, has significant potential for practical applications of the charged homopolypeptides. The findings raise attention especially to the pH fine tuning as an underappreciated control factor in surface modification and self-assembly.Peer reviewe

Ability of Poisson-Boltzmann Equation to Capture Molecular Dynamics Predicted Ion Distribution around Polyelectrolytes

Peer reviewe

Poly-L-arginine molecule properties in simple electrolytes : molecular dynamic modeling and experiments

Physicochemical properties of poly-L-arginine (P-Arg) molecules in NaCl solutions were determined by molecular dynamics (MD) modeling and various experimental techniques. Primarily, the molecule conformations, the monomer length and the chain diameter were theoretically calculated. These results were used to interpret experimental data, which comprised the molecule secondary structure, the diffusion coefficient, the hydrodynamic diameter and the electrophoretic mobility determined at various ionic strengths and pHs. Using these data, the electrokinetic charge and the effective ionization degree of P-Arg molecules were determined. In addition, the dynamic viscosity measurements for dilute P-Arg solutions enabledto determine the molecule intrinsic viscosity, which was equal to 500 and 90 for ionic strength of 10(−5) and 0.15 M, respectively. This confirmed that P-Arg molecules assumed extended conformations and approached the slender body limit at the low range of ionic strength. The experimental data were also used to determine the molecule length and the chain diameter, which agreed with theoretical predictions. Exploiting these results, a robust method for determining the molar mass of P-Arg samples, the hydrodynamic diameter, the radius of gyration and the sedimentation coefficient was proposed

Conformations of Poly- l -lysine Molecules in Electrolyte Solutions

Physicochemical properties of poly-l-lysine (PLL) hydrobromide were determined by molecular dynamics (MD) modeling and a variety of experimental techniques. Primarily, the density, the chain diameter, the monomer length, and the PLL molecule conformations were theoretically calculated. These results were applied for the interpretation of experimental data acquired for the PLL sample of average molar mass equal to 122 kg/mol. They comprised the diffusion coefficient, the hydrodynamic diameter, and the electrophoretic mobility of molecules determined for the ionic strength ranging from 2 × 10-5 to 0.15 M and pH 5.6. Using these data, the electrokinetic charge and the effective ionization degree of PLL molecules were determined as a function of ionic strength. Additionally, precise dynamic viscosity measurements for dilute PLL solutions were performed yielding the intrinsic viscosity, which decreased from 2420 to 120 for ionic strengths of 2 × 10-5 and 0.15 M, respectively. This confirmed that PLL molecules assume extended conformations in accordance with theoretical modeling. These data enabled to determine the molecule length, the chain diameter, and its effective molecule cross-section area for various ionic strengths. Therefore, it was concluded that the combined dynamic light scattering and viscosity measurements supplemented by MD modeling furnish reliable information about PLL macromolecule conformations in electrolyte solution. Besides the significance for basic science, the results obtained in this work can be exploited for precisely determining the molar mass of macroions.Peer reviewe

Hydration and Temperature Response of Water Mobility in Poly(diallyldimethylammonium)-Poly(sodium 4-styrenesulfonate) Complexes

The combination of all-atom molecular dynamics simulations with differential scanning calorimetry (DSC) has been exploited to investigate the influence of temperature and hydration on the water distribution and mobility in poly(diallyldimethylammonium) (PDADMA) and poly(sodium 4-styrenesulfonate) (PSS) complexes. The findings show that the vast majority of the water molecules hydrating the polyelectrolyte complexes (PECs) with 18-30 wt % hydration are effectively immobilized due to the strong interactions between the PE charge groups and water. Temperature and hydration were found to decrease similarly the fraction of strongly bound water. Additionally, at low hydration or at low temperatures, water motions become dominantly local vibrations and rotations instead of translational motion; translation dominance is recovered in a similar fashion by increase of both temperature and hydration. DSC experiments corroborate the simulation findings by showing that nonfreezing, bound water dominates in hydrated PECs atcomparable hydrations. Our results raise attention to water as an equal variable to temperature in the design and engineering of stimuli-responsive polyelectrolyte materials and provide mechanistic explanation for the similarity.Peer reviewe

Relaxation times of solid-like polyelectrolyte complexes of varying pH and water content

This work was supported by the National Science Foundation (grant no. 1905732) (J.L.L.), the National Science Centre, Poland (grant no. 2018/31/D/ST5/01866) (P.B.), and the Academy of Finland (project no. 309324) (M.S.). Computational resources by the PLGrid Infrastructure, Poland, CSC IT Centre for Science, Finland, and RAMI—RawMatTERS Finland Infrastructure are also gratefully acknowledged. M.S. is grateful for the support by the FinnCERES Materials Bioeconomy Ecosystem and use of the Bioeconomy Infrastructure at Aalto.The effect of complexation pH and water on the relaxation time and dynamics of polyelectrolyte (PE) complexes (PECs) and coacervates remains poorly understood. Here, we describe the dynamic mechanical behavior of solid-like PECs containing weak PEs poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) at varying complexation pH, relative humidity, and temperature with support from molecular dynamics simulations. Time–temperature, time–water, and time–intrinsic ion pair superposition principles are applied to obtain the relaxation times. It is shown that the natural log of relaxation time in hydrated PAH/PAA PECs is inversely proportional to the volume fraction of water (ln τ ∼ φw–1) for a given complexation pH. For all complexation pH values examined, the natural log of relaxation time collapsed into a single line when plotted against the ratio of the number of intrinsic ion pairs to water molecules (ln (τ) ∼ nintrinsic ion pairs/nwater). Taken together, this suggests that the relaxation of solid-like, hydrated PAH/PAA PECs is mediated by bound water at the intrinsic ion pair.Peer reviewe

SARS-CoV-2 Spike Protein (RBD) Subunit Adsorption at Abiotic Surfaces and Corona Formation at Polymer Particles

The adsorption kinetics of the SARS-CoV-2 spike protein subunit with the receptor binding domain at abiotic surfaces was investigated. A combination of sensitive methods was used such as atomic force microscopy yielding a molecular resolution, a quartz microbalance, and optical waveguide lightmode spectroscopy. The two latter methods yielded in situ information about the protein adsorption kinetics under flow conditions. It was established that at pH 3.5–4 the protein adsorbed on mica and silica surfaces in the form of compact quasi-spherical aggregates with an average size of 14 nm. The maximum coverage of the layers was equal to 3 and 1 mg m−2 at pH 4 and 7.4, respectively. The experimental data were successfully interpreted in terms of theoretical results derived from modeling. The experiments performed for flat substrates were complemented by investigations of the protein corona formation at polymer particles carried out using in situ laser Doppler velocimetry technique. In this way, the zeta potential of the protein layers was acquired as a function of the coverage. Applying the electrokinetic model, these primary data were converted to the dependence of the subunit zeta potential on pH. It was shown that a complete acid-base characteristic of the layer can be acquired only using nanomolar quantities of the protein

Time-Temperature and Time-Water Superposition Principles Applied to Poly(allylamine)/Poly(acrylic acid) Complexes

The dynamic mechanical and rheological behavior of polyelectrolyte coacervates and complex precipitates is of interest for many applications ranging from health to personal care. Hydration is an important factor, but its effect on the dynamic properties of polyelectrolyte complexes (PECs) is poorly understood. Here, we describe the dynamic behavior of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) complex precipitates at varying relative humidity values and temperatures using both dynamic mechanical analysis (DMA) and all-atom molecular dynamics simulations. To mirror the experimental system via simulation, the water content within the PEC is measured and used as the parameter of interest rather than relative humidity. In the experimental DMA, the modulus decreases with both increasing water content and temperature. The data are superimposed into a super master hydrothermal curve using the time-temperature superposition principle and the time-water superposition principle for the first time. The temperature-dependent shift factor (a T ) follows an Arrhenius relation, and the water-dependent shift factor (a W ) follows a log-linear relation with the water content in the complex. These results suggest that both temperature and water affect the dynamics of the PEC by similar mechanisms over the range investigated. All-atom molecular dynamics simulations show that an increase in the water content and temperature leads to similar changes in the polyelectrolyte chain mobility with little effect on the number of intrinsic ion pairs, suggesting the validity of time-water and time-temperature superposition principles.Peer reviewe

Comparing water-mediated hydrogen-bonding in different polyelectrolyte complexes

All-atom molecular dynamics simulations are used to investigate the polyelectrolyte-specific influence of hydration and temperature on water diffusion in hydrated polyelectrolyte complexes (PECs). Two model PECs were compared: poly(allylamine hydrochloride) (PAH)-poly(sodium 4-styrenesulfonate) (PSS) and poly(diallyldimethylammonium) (PDADMA)-poly(acrylic acid) (PAA). The findings show that the strength of the hydrogen bonding i.e. polyelectrolyte water interaction has enormous influence on the water mobility, which has implications for PEC structure and properties. A 10-fold difference in the average water diffusion coefficient between PAH-PSS and PDADMA-PAA PECs at the same hydration level is observed. The vast majority of the water molecules hydrating the PDADMA-PAA PECs, for hydrations in the range of 26-38 wt%, are effectively immobilized, whereas for PAH-PSS PECs the amount of immobilized water decreases with hydration. This points to the polyelectrolyte-specific character of the PE-water hydrogen bonding relationship with temperature. PAA-water hydrogen bonds are found to be significantly less sensitive to temperature than for PSS-water. The polyelectrolyte-water interactions, investigated via radial distribution function, hydrogen bond distance and angle distributions, are connected with resulting structure of the PECs. The PDADMA-PAA and PAH-PSS PECs are prepared experimentally and the states of water at different hydration levels is determined using differential scanning calorimetry (DSC). Experiments confirm the differences between PDADMA-PAA and PAH-PSS PECs observed in the theoretical modelling. The results suggest that the initial predictions of the PEC's bonding with water can be based on simple molecular-level considerations.Peer reviewe