Reduced Native State Stability in Crowded Cellular Environment Due to Protein–Protein Interactions

Abstract

The effect of cellular crowding environments on protein structure and stability is a key issue in molecular and cellular biology. The classical view of crowding emphasizes the volume exclusion effect that generally favors compact, native states. Here, results from molecular dynamics simulations and NMR experiments show that protein crowders may destabilize native states via protein-protein interactions. In the model system considered here, mixtures of villin head piece and protein G at high concentrations, villin structures become increasingly destabilized upon increasing crowder concentrations. The denatured states observed in the simulation involve partial unfolding as well as more subtle conformational shifts. The unfolded states remain overall compact and only partially overlap with unfolded ensembles at high temperature and in the presence of urea. NMR measurements on the same systems confirm structural changes upon crowding based on changes of chemical shifts relative to dilute conditions. An analysis of protein-protein interactions and energetic aspects suggests the importance of enthalpic and solvation contributions to the crowding free energies that challenge an entropic-centered view of crowding effects.

Residue level quantification of protein stability in living cells

“…4 and 5). Recent advances in both the experimental (5-9, 13, 14, 16-19, 24, 26, 56, 63) and theoretical (20)(21)(22)25) aspects of macromolecular crowding allow this stabilization to be rationalized in terms of the properties of GB1 and the E. coli cytoplasm.…”

“…Recent work has shown that the effects of macromolecular crowding on globular protein stability depend on the nature of the crowder (21,25,26,70). Synthetic polymers tend to act as inert spheres and are stabilizing.…”

“…Increased attention to transient, nonspecific protein-protein interactions (12)(13)(14)(15) has led both experimentalists (16)(17)(18)(19) and theoreticians (20)(21)(22) to recognize the effects of chemical interactions between crowder and test protein when assessing the net effect of macromolecular crowding. These weak, nonspecific interactions can reinforce or oppose the effect of hard-core repulsions, resulting in increased or decreased stability depending on the chemical nature of the test protein and crowder (23)(24)(25)(26).…”

Equilibrium simulation of trp-cage in the presence of protein crowders

“…[1][2][3][4][5] It is well established that excluded-volume interactions with crowder particles can have a significant stabilizing effect on globular proteins, as demonstrated by experiments with synthetic crowding agents such as Ficoll or dextran. 6 Today, experiments are increasingly often performed under more realistic crowding conditions, using concentrated protein solutions [7][8][9][10] or cells. 9,[11][12][13][14][15][16] In these systems, it has been shown that other interactions may dominate over the steric repulsions and lead to a net destabilization of globular proteins.…”

“…9,[11][12][13][14][15][16] In these systems, it has been shown that other interactions may dominate over the steric repulsions and lead to a net destabilization of globular proteins. [7][8][9][12][13][14]16 For a fundamental understanding of these effects, there is a need for computational approaches that permit the simulation of folding/unfolding equilibria in the presence of complex biomolecular crowding agents.…”