Wards in the keyway: amino acids with anomalous pK(a)s in calycins.

As a follow-up to our recent analysis of the electrostatics of bovine ß-lactoglobulin (Eberini et al. in Amino Acids 42:2019-2030, 2011), we investigated whether the occurrence in the native structure of calycins-the superfamily to which ß-lactoglobulin belongs-of amino acids with anomalous pK (a)s is an infrequent or, on the contrary, a common occurrence, and whether or not a general pattern may be recognized. To this aim, we randomly selected four calycins we had either purified from natural sources or prepared with recombinant DNA technologies during our previous and current structural and functional studies on this family. Their pIs vary over several pH units and their known functions are as diverse as carriers, enzymes, immunomodulators and/or extracellular chaperones. In our survey, we used both in silico prediction methods and in vitro procedures, such as isoelectric focusing, electrophoretic titration curves and spectroscopic techniques. By comparing the results under native conditions (no exposure of the proteins to chaotropic agents) to those after protein unfolding (in the presence of 8 M urea), a shift is observed in the pK (a) of at least one amino acid per protein, which results in a measurable change in pI. Three types of amino acids are involved: Cys, Glu, and His, their position varies along the calycin sequence. Although no common mechanism may thus be recognized, we hypothesize that the 'normalization' of anomalous pK (a)s may be the phenomenon that accompanies, and favors, structural rearrangements such as those involved in ligand binding by these proteins. An interesting, if anecdotal, validation to this view comes from the behavior of human retinol binding protein, for which the pI of the folded and liganded protein is intermediate between those of the folded and unliganded and of the unfolded protein forms. Likewise, both solid (from crystallography) and solution state (from CD spectroscopy) data confirm that the protein undergoes structural rearrangement upon retinol binding.

Electrostatics of folded and unfolded bovine ß-lactoglobulin.

We report on electrophoretic, spectroscopic, and computational studies aimed at clarifying, at atomic resolution, the electrostatics of folded and unfolded bovine ß-lactoglobulin (BLG) with a detailed characterization of the specific aminoacids involved. The procedures we used involved denaturant gradient gel electrophoresis, isoelectric focusing, electrophoretic titration curves, circular dichroism and fluorescence spectra in the presence of increasing concentrations of urea (up to 8 M), electrostatics computations and low-mode molecular dynamics. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility induced by urea are not just the result of changes in gyration radius upon unfolding. Electrophoretic titration curves run across a pH range of 3.5-9 in the presence of urea suggest that more than one aminoacid residue may have anomalous pKa value in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114, mainly due to changes in global and local desolvation. For His161, the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C terminus, the end-effect associated to the solvated form strongly influences its pKa value with extreme variation between crystal structures on one side and NMR or low-mode molecular dynamics structures on the other. The urea concentration effective in BLG unfolding depends on pH, with higher stability of the protein at lower pH.

Bacterial frataxin CyaY is the gatekeeper of iron-sulfur cluster formation catalyzed by IscS.

Frataxin is an essential mitochondrial protein whose reduced expression causes Friedreich's ataxia (FRDA), a lethal neurodegenerative disease. It is believed that frataxin is an iron chaperone that participates in iron metabolism. We have tested this hypothesis using the bacterial frataxin ortholog, CyaY, and different biochemical and biophysical techniques. We observe that CyaY participates in iron-sulfur (Fe-S) cluster assembly as an iron-dependent inhibitor of cluster formation, through binding to the desulfurase IscS. The interaction with IscS involves the iron binding surface of CyaY, which is conserved throughout the frataxin family. We propose that frataxins are iron sensors that act as regulators of Fe-S cluster formation to fine-tune the quantity of Fe-S cluster formed to the concentration of the available acceptors. Our observations provide new perspectives for understanding FRDA and a mechanistic model that rationalizes the available knowledge on frataxin.