Crowding-Induced Elongated Conformation of Urea-Unfolded Apoazurin: Investigating the Role of Crowder Shape in Silico

Abstract

Here, we show by solution nuclear magnetic resonance measurements that the urea-unfolded protein apoazurin becomes elongated when the synthetic crowding agent dextran 20 is present, in contrast to the prediction from the macromolecular crowding effect based on the argument of volume exclusion. To explore the complex interactions beyond volume exclusion, we employed coarse-grained molecular dynamics simulations to explore the conformational ensemble of apoazurin in a box of monodisperse crowders under strong chemically denaturing conditions. The elongated conformation of unfolded apoazurin appears to result from the interplay of the effective attraction between the protein and crowders and the shape of the crowders. With a volumeconserving crowder model, we show that the crowder shape provides an anisotropic direction of the depletion force, in which a bundle of surrounding rod-like crowders stabilize an elongated conformation of unfolded apoazurin in the presence of effective attraction between the protein and crowders.3

Small Neutral Crowding Solute Effects on Protein Folding Thermodynamic Stability and Kinetics

“…It was also observed that crowding conditions affect a protein structure, and a protein can even fold faster in an optimum concentration regime . Molecular crowders surrounding the protein in a cell have a high variability of sizes and shapes. , From a theoretical perspective, most computational and modeling studies assume molecular crowders similar to the protein size. , Such models allow an investigation in similar conditions found in vivo with a volume occupancy between 5% and 40%. , …”

Crowder-Induced Conformational Ensemble Shift in Escherichia coli Prolyl-tRNA Synthetase

“…The cytoplasm of cells such as Escherichia coli have a macromolecule concentration of up to 300-400 g/L, and 20-30% of the intracellular volume is occupied by macromolecules (9,10). Recent studies revealed that crowding affects protein stability (11)(12)(13)(14), folding (15,16), binding affinity (17,18), and catalysis (15,(19)(20)(21)(22)(23). Specifically, the size, structure, concentration, and chemical nature of crowding agents contribute to changes in stability, folding, conformational equilibrium, and catalysis when compared with dilute conditions (24)(25)(26)(27).…”

Modeling Crowded Environment in Molecular Simulations

“…However, recent studies have shown that such models are likely oversimplified. The shape of the crowders and the way they interact can influence the effects that the crowders exert on the molecules (O'Brien et al, 2011; Kang et al, 2015; Chen and Zhao, 2019; Zegarra et al, 2019), including how crowding affects protein diffusion (Balbo et al, 2013).…”

“…In a recent study (Zegarra et al, 2019), a set of crowders with different shapes was used to reproduce and explain an NMR experiment and show that the unfolded apoazurin protein becomes more extended upon addition of dextran crowders. In this work, spheres and spherocylinders of various lengths were used along with a CG protein model.…”