From catalyzing reactions to cell regulation, proteins represent one of the most important classes of complex molecules within living cells. Their folding and unfolding patterns from their primary to quaternary structures play a major role in determining their behavior and activity. The conditions that cause the misfolding of proteins also induce altered behavior in such molecules. One case in particular is the accumulation of toxic protein aggregates believed to affect the onset of neurodegenerative diseases such as Parkinson's and Huntington's. It has been shown that the three-dimensional structure of proteins is mainly dictated by their specific amino acid sequence. The transport properties and stability of proteins are, however, dependent on the local environment. Furthermore, while the behavior of such molecules is well understood under the bulk conditions, our understanding of their behavior under “crowded” cellular conditions is limited. The purpose of this study, therefore, is to determine the effects that high confinement representing a crowded medium, as may be seen in living cells, has on the structure, energy, and folding patterns of α-de novo-designed proteins for both attractive and repulsive nanopore walls.
Through the use of a united-atom force field the proteins are constructed by the protein intermediate resolution model (PRIME) via discontinuous molecular dynamics (DMD). DMD simulations have been carried out on the de novo-designed α-family of proteins with amino acid lengths of 9, 16, 23, and 30, with each amino acid chain consisting of 4 united atom beads. Three types of confined media have been considered: the space between a pair of flat walls, a cylindrical nanopore, and a spherical nanopore.
The DMD simulations for each amino acid length have been conducted using a range of nanopore sizes and temperatures for durations between 100 and 300 nanoseconds. The minimum nanopore radii wherein a fully folded stable protein can form in a slit and cylindrical nanopore are 1.28 and 1.31 nanometers, respectively, for all amino acid lengths. The respective values for a spherical nanopore are 1.91, 2.85, 3.87, and 4.88 nanometers for amino acid chain lengths of 9, 16, 23, and 30. The study conducted with a slit nanopore indicates that proteins with an α-helix native state are destabilized in the smallest nanopore size that does not affect the protein folding state. By slightly increasing the pore size, the optimal folding temperature is reached. Thus far, our studies indicate that increased confinement leads to enhanced instability with respect to both the folded and unfolded protein structures. We also show that for the α-helical proteins of various sizes in the cylindrical and spherical nanopores, the folding temperatures decrease with increased confinement for nanopore radii slightly larger than the minimum size. Furthermore, our present results reveal that in highly confined media the free energy surface becomes rough, and a new barrier for protein folding may appear close to the unfolded state.