We review what has been learned about the protein-folding problem from experimental kinetic studies. These studies reveal patterns of both great richness and surprising simplicity. The patterns can be interpreted in terms of proteins possessing an energy landscape which is largely, but not completely, funnel-like. Issues such as speed limitations of folding, the robustness of folding, the origin of barriers and cooperativity and the ensemble nature of transition states, intermediate and traps are assessed using the results from several experimental groups highlighting energy-landscape ideas as an interpretive framework.

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...

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease afflicting the voluntary motor system. More than 100 different mutations in the ubiquitously expressed enzyme superoxide dismutase-1 (SOD1) have been associated with the disease. To search for the nature of the cytotoxicity of mutant SOD1s, amounts, enzymic activities and structural properties of the protein as well as the CNS histopathology were examined in multiple transgenic murine models. In order to generate the ALS phenotype within the short lifespan of the mouse, more than 20-fold increased rates of synthesis of mutant SOD1s appear to be required. The organs of transgenic mice expressing human wild-type SOD1 or either of the G93A and D90A mutant proteins showed high steady-state protein levels. The major proportion of these SOD1s in the CNS were inactive due to insufficient Cu charging and all contained subfractions with a reduced C57-C146 intrasubunit disulphide bond. Both G85R and the truncated G127insTGGG mutant showed low steady-state protein levels, lacked enzyme activity and had no C57-C146 disulphide bond. These mutants were also enriched in the CNS relative to other organs, suggesting inefficient recognition and degradation of misfolded disulphide-reduced SOD1 in susceptible tissues. In end-stage disease, despite 35-fold differences in levels of mutant SOD1s, similar amounts of detergent-resistant aggregates accumulated in the spinal cord. Small granular as well as larger more diffuse human SOD1 (hSOD1)-inclusions developed in all strains, the latter more pronounced in those with high hSOD1 levels. Widespread vacuolizations were seen in the strains with high levels of hSOD1 but not those with low, suggesting these alterations to be artefacts related to high hSOD1 levels and not to the ALS-causing cytotoxicity. The findings suggest that the motoneuron degeneration could be due to long-term exposure to misfolded aggregation-prone disulphide-reduced SOD1, which constitutes minute subfractions of the stable mutants and larger proportions of the unstable mutants.

Although the rates of chemical reactions become faster with increasing temperature, the converse may be observed with protein-folding reactions. The rate constant for folding initially increases with temperature, goes through a maximum, and then decreases. The activation enthalpy is thus highly temperature dependent because of a large change in specific heat (delta Cp). Such a delta Cp term is usually presumed to be a consequence of a large decrease in exposure of hydrophobic surfaces to water as the reaction proceeds from the denatured state to the transition state for folding: the hydrophobic side chains are surrounded by "icebergs" of water that melt with increasing temperature, thus making a large contribution to the Cp of the denatured state and a smaller one to the more compact transition state. The rate could also be affected by temperature-induced changes in the conformational population of the ground state: the heat required for the progressive melting of residual structure in the denatured state will contribute to delta Cp. By examining two proteins with different refolding mechanisms, we are able to find both of these two processes; barley chymotrypsin inhibitor 2, which refolds from a highly unfolded state, fits well to a hydrophobic interaction model with a constant delta Cp of activation, whereas barnase, which refolds from a more structured denatured state, deviates from this ideal behavior.

The molecular mechanism by which the homodimeric enzyme Cu͞Zn superoxide dismutase (SOD) causes neural damage in amytrophic lateral sclerosis is yet poorly understood. A striking, as well as an unusual, feature of SOD is that it maintains intrasubunit disulfide bonds in the reducing environment of the cytosol. Here, we investigate the role of these disulfide bonds in folding and assembly of the SOD apo protein (apoSOD) homodimer through extensive protein engineering. The results show that apoSOD folds in a simple three-state process by means of two kinetic barriers: 2Dº2MºM 2. The early predominant barrier represents folding of the monomers (M), and the late barrier the assembly of the dimer (M 2). Unique for this mechanism is a dependence of protein concentration on the unfolding rate constant under physiological conditions, which disappears above 6 M Urea where the transition state for unfolding shifts to first-order dissociation of the dimer in accordance with Hammond-postulate behavior. Although reduction of the intrasubunit disulfide bond C57-C146 is not critical for folding of the apoSOD monomer, it has a pronounced effect on its stability and abolishes subsequent dimerization. Thus, impaired ability to form, or retain, the C57-C146 bond in vivo is predicted to increase the cellular load of marginally stable apoSOD monomers, which may have implications for the amytrophic lateral sclerosis neuropathology.protein folding ͉ protein stability ͉ disulfide bond ͉ transition-state shifts ͉ protein engineering T he mechanism by which mutant superoxide dismutase (SOD) leads to neural damage in the familial form of amyotrophic lateral sclerosis (ALS) is yet unknown (1-3). In analogy with other neurodegenerative disorders (4), however, an increasing body of observations (5-16) suggest that the ALS disease mechanism is coupled to destabilization or misfolding of the SOD structure (17), manifested ultimately by cellular inclusions of SOD aggregates (3,18). From a strictly energetic perspective, the native SOD structure is distinctive by containing one oxidized disulfide bond per monomer (C57-C146) in the reducing environment of the cytosol (19) (Fig. 1). Normally, disulfide bonds are maintained only under oxidizing conditions in the extracellular space (20, 21) where they increase protein stability by confining the configurational entropy of the denatured ensemble (22). Indications that the integrity of the C57-C146 bond may have bearing on the molecular events in ALS were recently provided by Tiwari and Hayward (15), who demonstrated that ALS-associated SOD mutants are more susceptible to chemical disulfide reduction than the wild-type protein. More detailed elucidation of the structural and energetic effects accompanying mutational perturbations and disulfide reduction has so far been prevented by scarce knowledge about how the SOD homodimer folds (23).In this study, we shed further light on this issue by mapping out the folding and assembly reaction of metal-depleted SOD through kinetic and thermodynamic analysis of...