Evaluation of the physical properties of experimental macromolecular crowding systems

Biological cells are crowded environments consisting of both large and small molecules. The macromolecular crowding observed in biological cells is likely very important to the structure and function of a living cell. Since the macromolecules can interact with each other, this makes the system complex and there remain several open questions. In the laboratory, artificial crowder molecules can be used to create experimental model systems that mimic the cellular environment. Artificial crowders such as the polysaccharide, Ficoll, have long been assumed to be compact and colloidal, but a holistic understanding of its structure and dynamics is lacking. This thesis investigates the structure of Ficoll using multiple experimental techniques. We report rheology, small angle neutron scattering, self diffusion and relaxation measurements using nuclear magnetic resonance experiments on two widely used artificial crowder molecules, Ficoll-70 and Ficoll-400. Our results, combining measures of structure, diffusion, relaxation and rheology, show that Ficolls are more polymer like than colloid like. Importantly, we find that the self-diffusion of HDO molecules in the suspension is an efficient probe to evaluate volume occupancy of the suspension under investigation. We then evaluate the physical properties of a protein crowder solution, BSA, and phytoglycogen, a natural plant based dendrimer using the methods developed to evaluate the Ficoll suspension properties. For all crowders, we find that the self diffusion coefficient decreases exponentially with a characteristic concentration of 10-12 wt %. We also observe that the NMR transverse relaxation of the solvent is a sensitive, independent measure of water confinement, which can be correlated with suspension rheology and self diffusion. To summarize, the highlight of this thesis is that structural and dynamical methods that report on macromolecules as well as solvent can provide a more complete view of macromolecular crowding

Concentration Dependence of Elastic and Viscoelastic Properties of Aqueous Solutions of Ficoll and Bovine Serum Albumin by Brillouin Light Scattering Spectroscopy

The cellular environment is crowded with macromolecules of different shapes and sizes. The effect of this macromolecular crowding has been studied in a variety of synthetic crowding environments: two popular examples are the compact colloid-like Ficoll macromolecule, and the globular protein bovine serum albumin (BSA). Recent studies have indicated a significant component of bound or surface-associated water in these crowders reduces the available free volume. In this work, Brillouin light scattering experiments were performed on aqueous solutions of Ficoll 70 and Ficoll 400 with concentrations ranging from 1 wt% to 35 wt% and BSA with concentrations of 1 wt% to 27 wt%. From the dependence of spectral peak parameters on polymer concentration, we determined fundamental solution properties: hypersound velocity, adiabatic bulk modulus and compressibility, apparent viscosity, and hypersound attenuation. Existing theory that ignores intermolecular interactions can only capture the observed linear trends in the frequency shift up to a threshold concentration, beyond which a quadratic term accounting for intermolecular interactions is necessary. This likely indicates a transition from the dilute to semi-dilute regime. In the Ficoll solutions (but not BSA) we see evidence for a central mode, with a characteristic relaxation time of 20 ps, that we attribute to exchange of the bound water.Comment: 10 pages, 4 figures, 4 table

Concentration Dependence of Elastic and Viscoelastic Properties of Aqueous Solutions of Ficoll and Bovine Serum Albumin by Brillouin Light Scattering Spectroscopy

The cellular environment is crowded with macromolecules of different shapes and sizes. The effect of this macromolecular crowding has been studied in a variety of synthetic crowding environments: two popular examples are the compact colloid-like Ficoll macromolecule and the globular protein bovine serum albumin (BSA). Recent studies have indicated that a significant component of bound or surface-associated water in these crowders reduces the available free volume. In this work, Brillouin light scattering experiments were performed on aqueous solutions of Ficoll 70 and Ficoll 400 with concentrations ranging from 1 to 35 wt % and BSA with concentrations of 1 to 27 wt %. From the dependence of spectral peak parameters on polymer concentration, we determined fundamental solution properties: hypersound velocity, adiabatic bulk modulus and compressibility, apparent viscosity, and hypersound attenuation. The existing theory that ignores intermolecular interactions can capture only the observed linear trends in the frequency shift up to a threshold concentration, beyond which a quadratic term accounting for intermolecular interactions is necessary. This likely indicates a transition from the dilute to semidilute regime. In the Ficoll solutions (but not BSA), we see evidence for a central mode, which is indicative of relaxation in the hydration shell of Ficoll

Concentration Dependence of Elastic and Viscoelastic Properties of Aqueous Solutions of Ficoll and Bovine Serum Albumin by Brillouin Light Scattering Spectroscopy

The cellular environment is crowded with macromolecules of different shapes and sizes. The effect of this macromolecular crowding has been studied in a variety of synthetic crowding environments: two popular examples are the compact colloid-like Ficoll macromolecule and the globular protein bovine serum albumin (BSA). Recent studies have indicated that a significant component of bound or surface-associated water in these crowders reduces the available free volume. In this work, Brillouin light scattering experiments were performed on aqueous solutions of Ficoll 70 and Ficoll 400 with concentrations ranging from 1 to 35 wt % and BSA with concentrations of 1 to 27 wt %. From the dependence of spectral peak parameters on polymer concentration, we determined fundamental solution properties: hypersound velocity, adiabatic bulk modulus and compressibility, apparent viscosity, and hypersound attenuation. The existing theory that ignores intermolecular interactions can capture only the observed linear trends in the frequency shift up to a threshold concentration, beyond which a quadratic term accounting for intermolecular interactions is necessary. This likely indicates a transition from the dilute to semidilute regime. In the Ficoll solutions (but not BSA), we see evidence for a central mode, which is indicative of relaxation in the hydration shell of Ficoll

Is Ficoll a Colloid or Polymer? A Multitechnique Study of a Prototypical Excluded-Volume Macromolecular Crowder

The in-cell environment is crowded with macromolecules, and the consequent reduction in free volume, based on the hard-sphere paradigm, is central to understanding macromolecular motions. A much-used model crowder, Ficoll, often assumed to be a compact, if not rigid, colloidal particle, is studied by rheology, small-angle neutron scattering, nuclear magnetic resonance diffusometry, and relaxometry. We find that the Ficoll suspension viscosity scales linearly with concentration cF in the dilute limit and as ∼cF3.8 at high cF, i.e, consistent with the 15/4 (de Gennes) scaling for a reptating polymer. The form factor of Ficoll, obtained via small-angle neutron scattering (SANS), resembles either a Gaussian polymer or a soft polymer blob. From NMR diffusion measurements, we obtain an effective volume fraction for Ficoll that accounts for Ficoll-bound water in two ways and show that each results in a volume occupancy of 60% to 70% in the crowding limit, much larger than the traditionally reported values of around 30%. If we persist with the colloid paradigm and examine the dependence of the zero-q structure factor obtained via SANS in terms of this effective volume fraction, we find that only a combination of particle softness and interparticle attractions, quantified using a computational model, can replicate the experimental S(0). The stark difference between effective and traditional volume occupancies affects the interpretation of previous experiments on macromolecular crowding and might explain the intriguing non-monotonicity observed in the dependence of protein relaxation rates on crowder concentration