Macromolecular dynamics and structure in crowded and confined environments

Macromolecular crowding and confinement, the effects caused by high concentrations of macromolecules in solution and/or in small compartments, are believed to influence diffusion processes, intermolecular interactions, protein folding, and intracellular transport in living cells. Understanding mechanisms of transport in biological systems (such as living cells) is complex and challenging. We construct cell mimetic environments in which the artificial macromolecules (e.g. polyethylene glycol, Ficoll70) are compartmentalized not in cells but in concentrated environments and agarose gel networks. In this work we have established a system to generate stable and monodisperse droplets of hierarchical confinement. The goal of this study is to measure translational diffusion in crowded and confined geometries of varying concentrations of different macromolecules on diffusion. We have combined the use of pulsed-fieldgradient nuclear magnetic resonance (PFG NMR) with small-angle neutron scattering (SANS) in order to obtain new insights in simple model systems of macromolecular crowding. The NMR and SANS techniques complement each other. Using PFG NMR technique, we have monitored the dynamics of synthetic macromolecules with multiple chemical components in complex environments. SANS, on the other hand, yields structure (size) of macromolecules. Our experimental findings in cell mimetic environments provide an important step towards gaining further insights into the effects of macromolecular crowding on diffusion and conformation

Polymer conformation and dynamics in crowded environments: A combined diffusion NMR and small-angle neutron scattering study

The effect of particles on the behavior of polymers in solution is important in a number of important phenomena such as the effect of “crowding” proteins in cells, colloid-polymer mixtures, and nanoparticle “fillers” in polymer solutions and melts. In this talk, I will present a study of the effect of spherical inert nanoparticles (which we refer to as “crowders”) on the diffusion coefficient and radius of gyration of polymers in solution using pulsed-field-gra- dient NMR and small-angle neutron scattering (SANS), respectively. In addition, the role of enthalpic crowder- crowder interactions on the crowding process is unknown: we can control this by varying charge on the crowder particle. Below a characteristic polymer concentration, which we identify as the overlap threshold concentration c⋆, the diffusion coefficients exhibit a plateau. Above c⋆, in a crossover region between the dilute and semidilute regimes, the (long-time) self-diffusion coefficients are found, universally, to decrease exponentially with polymer concentration at all crowder packing fractions, consistent with a structural basis for the long-time dynamics. When the polymer radius of gyration and crowder size are comparable, the polymer size is very weakly affected by the presence of crowders, consistent with recent computer simulations. We find that crowder charge only weakly affects polymer size and dynamics in the crowding limit, but that local macromolecular mobility depends strongly on molecular flexibility

Combining Diffusion NMR and Small-Angle Neutron Scattering Enables Precise Measurements of Polymer Chain Compression in a Crowded Environment

The effect of particles on the behavior of polymers in solution is important in a number of important phenomena such as the effect of “crowding” proteins in cells, colloid-polymer mixtures, and nanoparticle “fillers” in polymer solutions and melts. In this Letter, we study the effect of spherical inert nanoparticles (which we refer to as “crowders”) on the diffusion coefficient and radius of gyration of polymers in solution using pulsed-field-gradient NMR and small-angle neutron scattering (SANS), respectively. The diffusion coefficients exhibit a plateau below a characteristic polymer concentration, which we identify as the overlap threshold concentration c⋆. Above c⋆, in a crossover region between the dilute and semidilute regimes, the (long-time) self-diffusion coefficients are found, universally, to decrease exponentially with polymer concentration at all crowder packing fractions, consistent with a structural basis for the long-time dynamics. The radius of gyration obtained from SANS in the crossover regime changes linearly with an increase in polymer concentration, and must be extrapolated to c⋆ in order to obtain the radius of gyration of an individual polymer chain. When the polymer radius of gyration and crowder size are comparable, the polymer size is very weakly affected by the presence of crowders, consistent with recent computer simulations. There is significant chain compression, however, when the crowder size is much smaller than the polymer radius gyration

Frequency-dependent viscosity of salmon ovarian fluid has biophysical implications for sperm–egg interactions

Gamete-level sexual selection of externally fertilising species is usually achieved by modifying sperm behaviour with mechanisms that alter the chemical environment in which gametes perform. In fish, this can be accomplished through the ovarian fluid, a substance released with the eggs at spawning. While the biochemical effects of ovarian fluid in relation to sperm energetics have been investigated, the influence of the physical environment in which sperm compete remains poorly explored. Our objective was therefore to gain insights on the physical structure of this fluid and potential impacts on reproduction. Using soft-matter physics approaches of steady-state and oscillatory viscosity measurements, we subjected wild Atlantic salmon ovarian fluids to variable shear stresses and frequencies resembling those exerted by sperm swimming through the fluid near eggs. We show that this fluid, which in its relaxed state is a gel-like substance, displays a non-Newtonian viscoelastic and shear-thinning profile, where the viscosity decreases with increasing shear rates. We concurrently find that this fluid obeys the Cox–Merz rule below 7.6 Hz and infringes it above this level, thus indicating a shear-thickening phase where viscosity increases provided it is probed gently enough. This suggests the presence of a unique frequency-dependent structural network with relevant implications for sperm energetics and fertilisation dynamics

Pulsed field gradient NMR study of colloids

Pulsed field gradient nuclear magnetic resonance (NMR) experiments were performed on hard-sphere-like colloidal suspensions. We synthesized NMR-visible colloidal particles and measured spectrally resolved diffusion coefficients for monodisperse suspensions of different size particles. Results of these experiments show good agreement with theoretical expectation. We also probed a bidisperse (binary) colloidal suspension successfully and obtained the diffusion coefficients of two species simultaneously. The colloidal model system developed in this work will allow the study of colloidal phase behaviour in binary mixtures for different number and size ratios