Correlation of Levels of Folded Recombinant p53 in Escherichia coli with Thermodynamic Stability in Vitro

Abstract

The amount of folded functional protein in a cell is controlled by a number of factors, including the relative rates of its biosynthetic and specific degradation processes, and its intrinsic thermodynamic stability. Mutationinduced loss of stability is a common cause of disease. Many oncogenic mutants of the tumour suppressor p53, for example, reduce the intrinsic thermodynamic stability of the protein in vitro. We have analysed the level of recombinant folded human p53 core domain (p53C) and its mutants in Escherichia coli spanning a stability range of 6 kcal/mol to assess the effects of intrinsic thermodynamic stability in vivo in the absence of specific ubiquitin-mediated pathways in human cells. The levels of folded protein were measured fluorimetrically in living cells by fusing the gene of p53C upstream to that of green fluorescent protein and measuring the fluorescence relative to a control at various temperatures. At a fixed temperature, the amount of fluorescence is correlated with the thermodynamic stability of the mutant. The level of each protein varied with temperature according to a sigmoid curve that paralleled the melting in vitro, but the apparent T m was lower in vivo, because steady-state levels are observed rather than true thermodynamic equilibria. Our results show clearly that changes in the intrinsic thermodynamic stability of p53 reduce the level of folded and hence functional p53 substantially in E. coli, and provide insights into the correlation between protein instability and disease at the cellular level.

Metamorphic proteins mediate evolutionary transitions of structure

“…Similarly, the pI of Lib2-D2-15 dropped from to 8.3 to 7.0 in the evolved variant. However, although better expression in E. coli also correlates with increased thermodynamic stability (25,26), higher soluble expression of the pentameric variants was not accompanied by increased stability. Concomitantly, most of the mutations do not seem to affect the packing, and thereby stability, of the folded oligomeric forms.…”

“…The five subunits of the oligomeric lectins, although identical in sequence, adopt two, or even three, distinct structures and can thus exchange strands with up to four subunits in an asymmetric manner. Thus, more than one structure can be derived from the same sequence as previously noted (9,10,14,25,31,32). In the pentameric lectins, however, the different structures must coincide within the same oligomer to enable its assembly.…”

Thioflavin-S Staining of Bacterial Inclusion Bodies for the Fast, Simple, and Inexpensive Screening of Amyloid Aggregation Inhibitors

“…Interestingly, bacterial systems can be easily implemented to study the effect of genetic mutations [52][53][54][55] and/or the effects of drugs [56] on the aggregation of amyloid-prone proteins. We present here a protocol in which the direct determination of the Th-S relative fluorescence of bacterial cells allows to monitor changes in the intracellular aggregation of amyloidogenic proteins, without the requirement for expensive equipment.…”

Effects of Stability on the Biological Function of p53

“…1) were transiently expressed as a fusion protein with EGFP at the C terminus of p53 in H1299 cells. Fusion with EGFP does not affect the overall stability of p53, and EGFP fluoresces only when p53 is folded (26). This is based on the rapid protein-folding assay (27), in which an unfolded test protein fused to the N terminus of EGFP can interfere with the folding of EGFP.…”

“…The level of expression of the mutant p53 core domains in Escherichia coli, in the absence of Hdm2 and other E3 ligases feedback loops that are found in mammalian cells, depend on their thermodynamic stability (26). Destabilized mutants reach lower levels, since their rates of degradation are higher than that of stabilized mutants, although they all have the same rate of biosynthesis.…”