To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.

In order to use results from calorimetry or thermal unfolding curves to estimate the free energy change for protein unfolding at 25 degrees C, it is necessary to know the change in heat capacity for unfolding, delta Cp. We describe a new method for measuring delta Cp which is based on results from urea and thermal unfolding curves but does not require a calorimeter. We find that delta Cp = 1650 +/- 200 cal/(deg.mol) for the unfolding of ribonuclease T1 and that delta Cp = 2200 +/- 300 cal/(deg.mol) for the unfolding of ribonuclease A.

Background: MGP inhibits tissue calcification, but underlying mechanisms are understudied. Results: In MGP null mice, TG2 ablation prevents calcifying cartilaginous vascular lesions but does not affect elastocalcinosis and elastin fragmentation associated with increased elastase adipsin. Conclusion: MGP acts via two distinct mechanisms. Significance: Our study identifies TG2 and adipsin as potential therapeutic targets in vascular disease linked to MGP deficiency.

Transactive response DNA-binding protein 43 kDa (TDP-43) is an RNA transporting and processing protein whose aberrant aggregates are implicated in neurodegenerative diseases. The C-terminal domain of this protein plays a key role in mediating this process. However, the N-terminal domain (residues 1-77) is needed to effectively recruit TDP-43 monomers into this aggregate. In the present study, we report, for the first time, the essentially complete 1 H, 15N and 13C NMR assignments and the structure of the N-terminal domain determined on the basis of 26 hydrogen-bond, 60 torsion angle and 1058 unambiguous NOE structural restraints. The structure consists of an a-helix and six b-strands. Two b-strands form a b-hairpin not seen in the ubiquitin fold. All Pro residues are in the trans conformer and the two Cys are reduced and distantly separated on the surface of the protein. The domain has a well defined hydrophobic core composed of F35, Y43, W68, Y73 and 17 aliphatic side chains. The fold is topologically similar to the reported structure of axin 1. The protein is stable and no denatured species are observed at pH 4 and 25°C. At 4 kcalÁmol À1 , the conformational stability of the domain, as measured by hydrogen/deuterium exchange, is comparable to ubiquitin (6 kcalÁmol À1 ).The b-strands, a-helix, and three of four turns are generally rigid, although the loop formed by residues 47-53 is mobile, as determined by model-free analysis of the 15 N{ 1 H}NOE, as well as the translational and transversal relaxation rates.

A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis.