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We suggest that titin Ig domains in the I-band function as extensible components of muscle elasticity by stretching the hinge regions.

Protein unfolding can be induced both by heating and by cooling from ambient temperatures. 1 Accurate analysis of heat and cold denaturation processes has the potential to unveil hitherto obscure aspects of protein stability and dynamics. 2 For instance, while heat denaturation is generally highly cooperative, cold denaturation has been suggested to occur in a noncooperative fashion. 3,4 This view has been recently supported by an NMR study of ubiquitin in reverse micelles at very low temperatures, 5 but this is still controversial since Van Horn et al., 6 on the basis of similar NMR data, and Kitahara et al., 7 by an NMR study at 2 kbar, found a simple two-state behavior for the low-temperature unfolding of ubiquitin.To reach a consensus on this debate and other general issues, it is necessary to investigate cold denaturation further. However, since the cold denaturation of most proteins occurs well below the freezing point of water, full access to the cold denatured state is normally limited for the obvious reason that water freezes at 0 °C. The most common approach to circumvent this difficulty has been to try to raise the temperature of cold denaturation using destabilizing agents such as extreme pH values, chemical denaturants, cryosolvents, or very high pressure. 7-10 Alternatively, some laboratories used proteins destabilized by a combination of point mutations and denaturing agents. 9 The main drawback of these approaches is that it is not generally easy to extrapolate results to physiological conditions. On the other hand, there are methods aimed at keeping water in a supercooled condition, but these studies have also invariably used destabilized proteins. 11,12Following a different approach, we looked for a protein whose cold denaturation could be studied without the need for destabilization in a normal buffer at physiological pH within a temperature range accessible to several techniques. Here we describe the cold and heat denaturation of yeast frataxin (Yfh1) measured both by NMR and CD spectroscopies. In a systematic study of the factors that influence the thermal stability of the frataxin fold, we had previously shown that although they share the same fold, three orthologues from E. coli (CyaY), S. cerevisiae (Yfh1) and H. sapiens (hfra), are characterized, under the same conditions, by a remarkable variation of melting temperatures. 13 Yfh1, the one with lowest heat denaturation temperature, seemed a promising candidate for cold denaturation above 0 °C. Yfh1 and 15 Nlabeled Yfh1 were expressed in E. coli as described by He et al. 14 Since variations of ionic NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript strength lead to significant increases in the melting temperature, we restricted the present investigation to solutions of Yfh1 in salt-free buffers.We recorded 1D and 2D NMR spectra of Yfh1 either in TRIS at pH 7.0 or in HEPES at pH 7.0 in the temperature range −5 to 45 °C. Typically, 0.3-0.5 mM unlabeled or 15 N uniformly labeled protein samples were used. Thanks to t...

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.