Understanding the factors governing the thermal stability of proteins and correlating them to the sequence and structure is a complex and multiple problem that can nevertheless provide important information on the molecular forces involved in protein folding. Here, we have carried out a comparative genomic study to analyze the effects that different intrinsic and environmental factors have on the thermal stability of frataxins, a family of small mitochondrial iron-binding proteins found in organisms ranging from bacteria to humans. Low expression of frataxin in humans causes Friedreich's ataxia, an autosomal recessive neurodegenerative disease. The human, yeast, and bacterial orthologues were selected as representatives of different evolutionary steps. Although sharing high sequence homology and the same three-dimensional fold, the three proteins have a large variability in their thermal stabilities. Whereas bacterial and human frataxins are thermally stable, well-behaved proteins, under the same conditions yeast frataxin exists in solution as an unstable species with apprechable tracts in a conformational exchange. By designing suitable mutants, we show and justify structurally that the length of the C-terminus is an important intrinsic factor that directly correlates with the thermal stabilities of the three proteins. Thermal stability is also gained by the addition of Fe(2+). This effect, however, is not uniform for the three orthologues nor highly specific for iron: a similar albeit weaker stabilization is observed with other mono- and divalent cations. We discuss the implications that our findings have for the role of frataxins as iron-binding proteins.

CyaY is the bacterial ortholog of frataxin, a small mitochondrial iron binding protein thought to be involved in iron sulphur cluster formation. Loss of frataxin function leads to the neurodegenerative disorder Friedreich's ataxia. We have solved the solution structure of CyaY and used the structural information to map iron binding onto the protein surface. Comparison of the behavior of wild-type CyaY with that of a mutant indicates that specific binding with a defined stoichiometry does not require aggregation and that the main binding site, which hosts both Fe(2+) and Fe(3+), occupies a highly anionic surface of the molecule. This function is conserved across species since the corresponding region of human frataxin is also able to bind iron, albeit with weaker affinity. The presence of secondary binding sites on CyaY, but not on frataxin, hints at a possible polymerization mechanism. We suggest mutations that may provide further insights into the frataxin function.

Understanding the factors that determine protein stability is interesting because it directly reflects the evolutionary pressure coming from function and environment. Here, we have combined experimental and computational methods to study the stability of IscU, a bacterial scaffold protein highly conserved in most organisms and an essential component of the iron-sulfur cluster biogenesis pathway. We demonstrate that the effect of zinc and its consequence strongly depend on the sample history. IscU is a marginally stable protein at low ionic strength to the point that undergoes cold denaturation at around 28 C with a corresponding dramatic decrease of enthalpy, which is consistent with the fluxional nature of the protein. Presence of constitutively bound zinc appreciably stabilizes the IscU fold, whereas it may cause protein aggregation when zinc is added back posthumously. We discuss how zinc coordination can be achieved by different side chains spatially available and all competent for tetrahedral coordination. The individual absence of some of these residues can be largely compensated by small local rearrangements of the others. We discuss the potential importance of our findings in vitro for the function in vivo of the protein.

The taste of peptides is seldom one of the most relevant issues when one considers the many important biological functions of this class of molecules. However, peptides generally do have a taste, covering essentially the entire range of established taste modalities: sweet, bitter, umami, sour and salty. The last two modalities cannot be attributed to peptides as such because they are due to the presence of charged terminals and/or charged side chains, thus reflecting only the zwitterionic nature of these compounds and/or the nature of some side chains but not the electronic and/or conformational features of a specific peptide. The other three tastes, that is, sweet, umami and bitter, are represented by different families of peptides. This review describes the main peptides with a sweet, umami or bitter taste and their relationship with food acceptance or rejection. Particular emphasis will be given to the sweet taste modality, owing to the practical and scientific relevance of aspartame, the well-known sweetener, and to the theoretical importance of sweet proteins, the most potent peptide sweet molecules.

Protein unfolding occurs at both low and high temperatures, although in most cases, only the high-temperature transition can be experimentally studied. A pressing question is how much the low-and high-temperature denatured states, although thermodynamically equivalent, are structurally and kinetically similar. We have combined experimental and computational approaches to compare the high-and low-temperature unfolded states of Yfh1, a natural protein that, at physiologic pH, undergoes cold and heat denaturation around 0°C and 40°C without the help of ad hoc destabilization. We observe that the two denatured states have similar but not identical residual secondary structures, different kinetics and compactness and a remarkably different degree of hydration. We use molecular dynamics simulations to rationalize the role of solvation and its effect on protein stability.Crown