Macromolecular crowding studies of amino acids using NMR diffusion measurements and molecular dynamics simulations

Abstract

Molecular crowding occurs when the total concentration of macromolecular species in a solution is so high that a considerable proportion of the volume is physically occupied and therefore not accessible to other molecules. This results in significant changes in the solution properties of the molecules in such systems. Macromolecular crowding is ubiquitous in biological systems due to the generally high intracellular protein concentrations. The major hindrance to understanding crowding is the lack of direct comparison of experimental data with theoretical or simulated data. Self-diffusion is sensitive to changes in the molecular weight and shape of the diffusing species, and the available diffusion space (i.e., diffusive obstruction). Consequently, diffusion measurements are a direct means for probing crowded systems including the self-association of molecules. In this work, nuclear magnetic resonance (NMR) measurements of the self-diffusion of four amino acids (glycine, alanine, valine and phenylalanine) up to their solubility limit in water were compared directly with molecular dynamics simulations. The experimental data were then analyzed using various models of aggregation and obstruction. Both experimental and simulated data revealed that the diffusion of both water and the amino acids were sensitive to the amino acid concentration. The direct comparison of the simulated and experimental data afforded greater insights into the aggregation and obstruction properties of each amino acid.

Challenges and opportunities in connecting simulations with experiments via molecular dynamics of cellular environments

“…NMR experiments are especially suitable to cover a wide range of time scales [27, 28]. NMR data is often compared with molecular dynamics simulations because processes occurring on different time scales can be isolated and matched to the simulation time scales [29–32]; however, the standard interpretation of NMR data usually depends on certain assumptions that can become problematic in highly crowded cellular systems. Using dynamic relaxation of protein backbone amide N-H vectors as an example (see figure 2), the resulting dynamics is a result of combining internal dynamics with rotational and translational diffusion.…”

Crowding in Cellular Environments at an Atomistic Level from Computer Simulations

“…123 A meaningful comparison between experiments and simulations also requires long simulations as well as reliable force field parameters for solutes and solvent and faces potential experimental difficulties in analyzing highly retarded and likely complex dynamics in concentrated solutions, e.g., via nuclear magnetic resonance (NMR) spectroscopy. 81,175 …”

Physical characterization using diffusion NMR spectroscopy

“…Similarly, dNMR can be used to experimentally test theoretical models and molecular simulation results, particularly in biological systems . Aggregation of macromolecules is a particular biological process that can be modelled in silica with different degrees of reliability (depending on the model used for simulation), but as well can be probed with dNMR experimentally.…”

“…Blue—Lekkerkerker and Dhont, black—Han and Herzfeld, green—Tokuyama and Oppenheim (short interactions), red—Tokuyama and Oppenheim (long interactions), purple—Jönsson et al From Ref. with permission.…”

“…Similarly, dNMR can be used to experimentally test theoretical models and molecular simulation results, particularly in biological systems. [55,56] Aggregation of macromolecules is a particular biological process that can be modelled in silica with different degrees of reliability (depending on the model used for simulation), but as well can be probed with dNMR experimentally. Macromolecules (such as amino acids) exhibit aggregation and crowding [57,58] as their concentration in a solution increases the diffusive behaviour of such crowded macromolecules changes compared to the unrestricted diffusion in a solution.…”