Protein folding and assembly in confined environments: Implications for protein aggregation in hydrogels and tissues

Abstract

In the biological milieu of a cell, soluble crowding molecules and rigid confined environments strongly influence whether the protein is properly folded, intrinsically disordered proteins assemble into distinct phases, or a denatured or aggregated protein species is favored. Such crowding and confinement factors act to exclude solvent volume from the protein molecules, resulting in an increased local protein concentration and decreased protein entropy. A protein's structure is inherently tied to its function. Examples of processes where crowding and confinement may strongly influence protein function include transmembrane protein dimerization, enzymatic activity, assembly of supramolecular structures (e.g., microtubules), nuclear condensates containing transcriptional machinery, protein aggregation in the contexts of disease and protein therapeutics. Historically, most protein structures have been determined from pure, dilute protein solutions or pure crystals. However, these are not the environments in which these proteins function. Thus, there has been an increased emphasis on analyzing protein structure and dynamics in more "in vivo-like" environments. Complex in vitro models using hydrogel scaffolds to study proteins may better mimic features of the in vivo environment. Therefore, analytical techniques need to be optimized for real-time analysis of proteins within hydrogel scaffolds.

Impact of Four Common Hydrogels on Amyloid-β (Aβ) Aggregation and Cytotoxicity: Implications for 3D Models of Alzheimer’s Disease

“…Further discussion on hydrogels and protein aggregation may be found in our recent review article. 66 We were also interested in these hydrogels given their range of physiochemical properties and potential to interact with cells.…”

A first-takes-all model of centriole copy number control based on cartwheel elongation

“…Our model is not necessarily inconsistent with these results. Several other factors may contribute to increased stability of SAS-6 complexes in vivo , including other partner proteins, such as Cep135/Bld10p [ 30 ], the presence of scaffolds [ 36 ], molecular crowding [ 39 , 40 ], or cartwheel stacking itself. In addition, the aforementioned kinetic measurements were based on truncated SAS-6 N-termini, whose behaviour may differ from those of the full protein.…”

Combined Effects of Confinement and Macromolecular Crowding on Protein Stability

“…Research, both theoretical and experimental, over the past couple of decades has underscored the importance of replicating in vivo conditions to study protein structure and function [ 1 , 2 , 3 , 4 , 5 , 6 ]. Native cellular environments, such as the cytoplasm, are packed with biomacromolecules that subject proteins to highly confined and crowded environments, and these conditions have been shown to impact the equilibria of biochemical processes such as protein folding, as well as protein function and stability [ 2 , 3 ].…”

“…Until recently, most in vitro investigations of protein function and stability have been based on simple buffer systems that do not closely mimic the complex in vivo cellular environments [ 1 , 2 , 3 , 4 ]. Inside cells, proteins exist and function in highly crowded and compartmentalized environments that have a significant impact on several of their functions, including diffusion, enzymatic activity, protein–protein interactions, and folding, unfolding, and refolding [ 5 , 6 ]. Several studies have proposed the use of high concentrations of natural and synthetic macromolecules to study crowding [ 7 , 8 , 9 , 10 ], and encapsulating proteins within the pores of silica, polyacrylamide, or other hydrogels to study confinement [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ].…”