Joshua A. Riback

Reversible, Specific, Active Aggregates of Endogenous Proteins Assemble upon Heat Stress

Heat causes protein misfolding and aggregation and, in eukaryotic cells, triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying >170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degradation. Stable-isotope labeling reveals that even severely aggregated endogenous proteins are disaggregated without degradation during recovery from shock, contrasting with the rapid degradation observed for many exogenous thermolabile proteins. Although aggregation likely inactivates many cellular proteins, in the case of a heterotrimeric aminoacyl-tRNA synthetase complex, the aggregated proteins remain active with unaltered fidelity. We propose that most heat-induced aggregation of mature proteins reflects the operation of an adaptive, autoregulatory process of functionally significant aggregate assembly and disassembly that aids cellular adaptation to thermal stress.

Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water

An expanded view of disordered proteins Disordered proteins sample an ensemble of conformations, but it has remained unclear how compact these conformations are in water. Polymer physics relates the radius of gyration (Rg) to solvent quality, with more chain collapse occurring in poorer solvents. Riback et al. developed an analysis scheme that allows them to extract solvent quality and Rg from a single small-angle x-ray scattering measurement. Applying this method, they found that even disordered proteins with low net charge and high hydrophobicity remain expanded in water. Science, this issue p. 238 Intrinsically disordered proteins are often expanded in water, which affects their function, and dynamics and may protect them from aggregation. A substantial fraction of the proteome is intrinsically disordered, and even well-folded proteins adopt non-native geometries during synthesis, folding, transport, and turnover. Characterization of intrinsically disordered proteins (IDPs) is challenging, in part because of a lack of accurate physical models and the difficulty of interpreting experimental results. We have developed a general method to extract the dimensions and solvent quality (self-interactions) of IDPs from a single small-angle x-ray scattering measurement. We applied this procedure to a variety of IDPs and found that even IDPs with low net charge and high hydrophobicity remain highly expanded in water, contrary to the general expectation that protein-like sequences collapse in water. Our results suggest that the unfolded state of most foldable sequences is expanded; we conjecture that this property was selected by evolution to minimize misfolding and aggregation.

Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core

Significance The basis of protein-folding cooperativity and stability elicits a variety of opinions, as does the existence and importance of possible residual structure in the denatured state. We examine these issues in a protein that is striking in its dearth of hydrophobic burial and its lack of canonical α and β structures, while having a low sequence complexity with 46% glycine. Unexpectedly, the protein’s folding behavior is similar to that observed for typical globular proteins. This enigma forces a reexamination of the possible combination of factors that can stabilize a protein. The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

Response to Comment on “Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water”

Best et al. claim that we provide no convincing basis to assert that a discrepancy remains between FRET and SAXS results on the dimensions of disordered proteins under physiological conditions. We maintain that a clear discrepancy is apparent in our and other recent publications, including results shown in the Best et al. comment. A plausible origin is fluorophore interactions in FRET experiments.

Commonly-used FRET fluorophores promote collapse of an otherwise disordered protein.

The dimensions that unfolded and intrinsically disordered proteins (IDPs) adopt at low or no denaturant remains controversial. We recently developed an innovative analysis procedure for small-angle X-ray scattering (SAXS) profiles and found that even relatively hydrophobic IDPs remain nearly as expanded as the chemically denatured ensemble, rendering them significantly more expanded than is inferred from many fluorescence resonance energy transfer (FRET) studies. Here we show that fluor-phores typical of those added to IDPs for FRET studies contribute to this discrepancy. Specifically, we find that labeling a highly expanded IDP with Alexa488 causes its ensemble to contract significantly. We also tested the recent suggestion that FRET and SAXS results can be reconciled if, for unfolded proteins (and as opposed to the case for ideal random flight homopolymers), the radius of gyration (Rg) can vary independently from the chain’s end-to-end distance (Ree). Our analysis indicates, however, that SAXS is able to accurately extract Rg, ν and Ree even for heteropolymeric, protein-like sequences. From these studies we conclude that mild chain contraction and fluorophore-based interactions at lower denaturant concentrations, along with improved analysis procedures for both SAXS and FRET, can explain the preponderance of existing data regarding the nature of polypeptide chains unfolded in the absence of denaturant. Significance Statement Proteins can adopt a disordered ensemble, either prior to folding or as a part of their function. Simulations and fluorescence resonance energy transfer (FRET) studies often describe these disordered conformations as more compact than the fully random-coil state, whereas small-angle X-ray scattering studies (SAXS) indicate an expanded ensemble closely approximating the dimensions expected for the random coil. Resolving this discrepancy will enable more accurate predictions of protein folding and function. Here we reconcile these views by showing that the addition of common FRET fluorophores reduces the apparent dimensions of a disordered protein. Detailed analysis of both techniques, along with accounting for a moderate amount of fluorophore-induced contraction, demonstrates that disordered and unfolded proteins often remain well solvated and largely expanded in the absence of denaturant, properties that presumably minimize misfolding and aggregation.