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The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.

The folding of staphylococcal nuclease (SNase) is known to proceed via a major intermediate in which the central OB subdomain is folded and the C-terminal helical subdomain is disordered. To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail, using high-pressure NMR, the consequences of cavity creating mutations in each of the two subdomains of an ultrastable SNase, Δ+PHS. The stabilizing mutations of Δ+PHS enhanced the population of the major folding intermediate. Cavity creation in two different regions of the Δ+PHS reference protein, despite equivalent effects on global stability, had very distinct consequences on the complexity of the folding free energy landscape. The L125A substitution in the C-terminal helix of Δ+PHS slightly suppressed the major intermediate and promoted an additional excited state involving disorder in the N-terminus, but otherwise decreased landscape heterogeneity with respect to the Δ+PHS background protein. The I92A substitution, located in the hydrophobic OB-fold core, had a much more profound effect, resulting in a significant increase in the number of intermediate states and implicating the entire protein structure. Denaturant (GuHCl) had very subtle and specific effects on the landscape, suppressing some states and favoring others, depending upon the mutational context. These results demonstrate that disrupting interactions in a region of the protein with highly cooperative, unfrustrated folding has very profound effects on the roughness of the folding landscape, whereas the effects are less pronounced for an energetically equivalent substitution in an already frustrated region.

The time required to fold proteins usually increases significantly under conditions of high pressure. Taking advantage of this general property of proteins, we combined P-jump experiments with NMR spectroscopy to examine in detail the folding reaction of staphylococcal nuclease (SNase) and of some of its cavity-containing variants. The nearly 100 observables that could be measured simultaneously collectively describe the kinetics of folding as a function of pressure and denaturant concentration with exquisite site-specific resolution. SNase variants with cavities in the central core of the protein exhibit a highly heterogeneous transition-state ensemble (TSE) with a smaller solvent-excluded void volume than the TSE of the parent SNase. This heterogeneous TSE experiences Hammond behavior, becoming more native-like (higher molar volume) with increasing denaturant concentration. In contrast, the TSE of the L125A variant, which has a cavity at the secondary core, is only slightly different from that of the parent SNase. Because pressure acts mainly to eliminate solvent-excluded voids, which are heterogeneously distributed throughout structures, it perturbs the protein more selectively than chemical denaturants, thereby facilitating the characterization of intermediates and the consequences of packing on folding mechanisms. Besides demonstrating how internal cavities can affect the routes and rates of folding of a protein, this study illustrates how the combination of P-jump and NMR spectroscopy can yield detailed mechanistic insight into protein folding reactions with exquisite site-specific temporal information.

The redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing pH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation ( E 1 / 2 ) changed from − 65.3 to + 146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P 50 from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins.