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.

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Iron-sulfur clusters are widely represented in most organisms, but the mechanism of their formation is not fully understood. Of the two main proteins involved in cluster formation, NifS/IscS and NifU/IscU, only the former has been well studied from a structural point of view. Here we report an extensive structural characterization of Escherichia coli IscU. We show by a variety of physico-chemical techniques that E. coli IscU construct can be expressed to high purity as a monomeric protein, characterized by an ab fold with high a-helix content. The high melting temperature and the reversibility of the thermal unfolding curve (as measured by CD spectroscopy) hint at a well ordered stable fold. The excellent dispersion of cross peaks in the 1 H-15 N correlation spectrum is consistent with these observations. Monomeric E. coli IscU is able to provide a scaffold for Iron-sulfur cluster assembly, but has no direct interaction with either Fe(II) or Fe(III) ions, suggesting the need of further partners to achieve a stable interaction.

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum is synthesized by schizont stage parasites and has been implicated in merozoite invasion of host erythrocytes. Phage-display techniques have recently been used to identify two 15-residue peptides, F1 and F2, which bind specifically to P. falciparum AMA1 and inhibit parasite invasion of erythrocytes [Li, F., et al. (2002) J. Biol. Chem. 277, 50303-50310]. We have synthesized F1, F2, and three peptides with high levels of sequence identity, determined their relative binding affinities for P. falciparum AMA1 with a competition ELISA, and investigated their solution structures by NMR spectroscopy. The strongest binding peptide, F1, contains a beta-turn that includes residues identified via an alanine scan as being critical for binding to AMA1 and inhibition of merozoite invasion of erythrocytes. The three F1 analogues include a 10-residue analogue of F1 truncated at the C-terminus (tF1), a partially scrambled 15-mer (sF1), and a disulfide-constrained 14-mer (F1tbp) which is related to F1 but has a sequence identical to that of a disulfide-constrained loop in the first epidermal growth factor module of the latent transforming growth factor-beta binding protein. tF1 and F1tbp bound competitively with F1 to AMA1, and all three contain a type I beta-turn encompassing key residues involved in F1 binding. In contrast, sF1 lacked this structural motif, and did not compete for binding to AMA1 with F1; rather, sF1 contained a type III beta-turn involving a different part of the sequence. Although F2 was able to bind to AMA1, it was unstructured in solution, consistent with its weak invasion inhibitory effects. Thus, the secondary structure elements observed for these peptides in solution correlate well with their potency in binding to AMA1 and inhibiting merozoite invasion. The structures provide a valuable starting point for the development of peptidomimetics as antimalarial antagonists directed at AMA1.