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A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.

Background: Ubiquitination of intracellular N termini lysines inhibits cleavage/stimulation of ␣,␤,␥ epithelial sodium channel (ENaC) extracellular domains. Results: The N terminus of ␥-ENaC attains secondary structure upon sensing membrane phospholipids. Basic residues promote ENaC cleavage. Conclusion: Ubiquitination obscures residues mediating allosteric stimulatory N-terminal structural transition that promotes ENaC cleavage/stimulation. Significance: This study presents a new mechanism of allosteric linkage between cytosolic signaling pathways and extracellular ENaC proteolysis.

Isotope-edited infrared (IR) spectroscopy is a powerful tool for studying structural and dynamical properties of peptides and proteins with site-specific resolution. Labeling of selected amide carbonyls with (13)C results in detectable sidebands of amide I' vibrations, which provide information about local conformation and/or solvent exposure without structural perturbation to the protein. Incorporation of isotopically labeled amino acids at specific positions is achieved by the chemical synthesis of the studied proteins. We describe the basic procedures for synthesis of (13)C isotopically edited protein samples, experimental IR spectroscopic measurements, and analysis of the site-specific structural changes from the thermal unfolding IR data.