Specific Interactions and Environment Flexibility Tune Protein Stability under Extreme Crowding

Abstract

Macromolecular crowding influences protein mobility and stability in vivo. A precise description of the crowding effect on protein thermal stability requires the estimate of the combined effects of excluded volume, specific protein−environment interactions, as well as the thermal response of the crowders. Here, we explore an ideal model system, the lysozyme protein in powder state, to dissect the factors controlling the melting of the protein under extreme crowding. By deploying state-of-the art molecular simulations, supported by calorimetric experiments, we assess the role of the environment flexibility and of intermolecular electrostatic interactions. In particular, we show that the temperature-dependent flexibility of the macromolecular crowders, along with specific interactions, significantly alleviates the stabilizing contributions of the static volume effect.

Computational Insights into the Unfolding of a Destabilized Superoxide Dismutase 1 Mutant

“…The a99SB-disp force field also exhibited a higher residual secondary structure content at high temperatures (see Figure 2B). It should be noted that both force fields significantly overestimated the melting temperatures (see Figure 2), a phenomenon that we previously saw in REST2 simulations of a protein in dilute [33] and crowded solutions [26,35], as well as in powder [34,36] and that has commonly been reported for protein force fields [49]. This upshift in the observed melting temperature was particularly marked for a99SB-disp.…”

“…Compared to REMD, REST2 uses a Hamiltonian-rescaling procedure to selectively heat protein degrees of freedom, which results in a substantial reduction in the number of replicas required to sample the conformational landscape of the given system [ 27 ]. We demonstrated previously the effectiveness of REST2 at reproducing thermal stability trends for small and midsize proteins in dilute- [ 28 , 33 ] and crowded conditions [ 26 , 34 , 35 , 36 ].…”

Correlating the Local and Global Dynamics of an Enzyme in the Crowded Milieu

“…The motions in this range can be referred to as the intermediate time-scale motions of the protein, which are largely attributed to chain segment motions. 1,21 The flexibility of the crowders also increases with increasing temperature, 41 thereby bringing about a drop in the average solvation time. An examination of the three-dimensional crystal structure of the protein shows that Cys 132, located in the loop region, is quite exposed to the bulk solvent and relatively free when compared to the probe attached at positions Cys-74 and Cys-209.…”

Macromolecular crowding: how shape and interaction affect the structure, function, conformational dynamics and relative domain movement of a multi-domain protein

“…4–26 At present, the general consensus is that every biological phenomenon (like stability, dynamics, activity, folding, aggregation, association, binding, cell culture, tissue engineering, and so on) is modulated in a crowded environment. 4–27 Given that performing an experiment and data analysis in a living cell or cell lysate is extremely challenging, most of the studies involve small co-solutes (that probably represent natural osmolytes) and large synthetic macromolecules (like dextran, Ficoll, and polyethylene glycol (PEG) that represents bio-macromolecules like proteins and nucleic acids) as the crowding agents. 4–27 Though natural proteins could be a better candidate and physiologically more relevant as a macromolecular crowder, they cannot be used in a systematic study ( ca.…”

“…4–27 Given that performing an experiment and data analysis in a living cell or cell lysate is extremely challenging, most of the studies involve small co-solutes (that probably represent natural osmolytes) and large synthetic macromolecules (like dextran, Ficoll, and polyethylene glycol (PEG) that represents bio-macromolecules like proteins and nucleic acids) as the crowding agents. 4–27 Though natural proteins could be a better candidate and physiologically more relevant as a macromolecular crowder, they cannot be used in a systematic study ( ca. size/shape effect) to decipher the mechanism.…”