Thermodynamics of protein destabilization in live cells

Abstract

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37°C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.thermodynamics | protein stability | crowding | in vivo | NMR U nlike their static impression in X-ray structures and textbook illustrations, some proteins are tuned to work at marginal structural stability. The advantage of such tuning is that it enables the protein to easily switch from one conformation to another, providing sensitive functional control. A well-known example is the tumor suppressor P53 whose function in gene regulation relies on a complex interplay of local folding-unfolding transitions (1). Likewise, the maturation pathway of the radical scavenger Cu/Zn superoxide dismutase (SOD1) involves a marginally stable apo species that seems required for interorganelle trafficking (2) and effective chaperone-assisted metal loading (3). As an inevitable consequence of such near-equilibrium action, however, the proteins become critically sensitive to perturbations (1): Mutation of SOD1 triggers with full penetrance late-onset neurodegenerative disease even though the causative mutations shift the structural equilibrium only by less than a factor of 3 (4). In the latter case, it is not the loss of native function that poses the acute problem, but rather the promotion of competing disordered SOD1 conformations that eventually exhaust the cellular proteostasis system and end up in pathologic deposits (5-8). Uncovering the rules, capacity and limitations of this delicate interplay between individual proteins and the cellular components (9, 10) requires not only information about the in vivo response to molecular perturbations, but also precise quantification of the structural equilibria at play. The question is then, to what extent are existing data obtained under simplified conditions in vitro transferable to the complex environment in live cells (11)? The answer is not clear cut. Defying predictions from steric crowding effects (11-13), experimental data have shown that cells in some cases stabilize (14-19) and in other cases destabilize (20-25) the native protein structures. In this study, we shed light on these s...

In-cell thermodynamics and a new role for protein surfaces

“…1C). T m and ΔG°′ U either decreased or were unchanged compared with buffer (SI Appendix, Tables S1 and S2), consistent with other studies, (13,(15)(16)(17)(18) but inconsistent with previously accepted crowding theory (1,2). ΔC°′ p,U is the same in buffer and in cells (SI Appendix, Table S1).…”

“…This approach probably overestimates ΔG°′ U, T (gives the maximum stability, "cells-corrected" in Fig. 1C) because in vitro studies with protein crowders as well as in-cell studies show that destabilizing weak attractive interactions often dominate stabilizing hard-core excluded volume effect (13)(14)(15). We expect the true ΔG°′ U, T lies between the two values.…”

Protein dynamics and function from solution state NMR spectroscopy

“…inside living cells. 'In-cell' and 'in-lysate' NMR spectroscopy (Danielsson et al 2013(Danielsson et al , 2015Serber et al 2005Serber et al , 2007Smith et al 2013) have developed to a point where new aspects of protein function can be revealed (Banci et al 2013). For instance in a recent study Selenko and co-workers developed an NMR-based methodology that enables quantification of the temporal phosphorylation and de-phosphorylation of short protein segments in cell lysates (Thongwichian et al 2015).…”

Markov modeling of peptide folding in the presence of protein crowders

“…[1][2][3] To understand the role of other interactions, in recent years, there have been increasing efforts to perform computer simulations with realistic crowder molecules, 4-11 rather than hard-sphere crowders. When analyzing these large systems, a major challenge lies in identifying the main states and dynamical modes, which may not be easily anticipated.…”