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By performing differential scanning calorimetry(DSC) measurements on RNase A, we studied the stabilization provided by the addition of potassium aspartate(KAsp) or potassium glutamate (KGlu) and found that it leads to a significant increase in the denaturation temperature of the protein. The stabilization proves to be mainly entropic in origin. A counteraction of the stabilization provided by KAsp or KGlu is obtained by adding common denaturants such as urea, guanidinium chloride, or guanidinium thiocyanate. A rationalization of the experimental data is devised on the basis of a theoretical approach developed by one of the authors. The main contribution to the conformational stability of globular proteins comes from the gain in translational entropy of water and co-solute ions and/or molecules for the decrease in solvent-excluded volume associated with polypeptide folding (i.e., there is a large decrease in solvent-accessible surface area). The magnitude of this entropic contribution increases with the number density and volume packing density of the solution. The two destabilizing contributions come from the conformational entropy of the chain, which should not depend significantly on the presence of co-solutes, and from the direct energetic interactions between co-solutes and the protein surface in both the native and denatured states. It is the magnitude of the latter that discriminates between stabilizing and destabilizing agents.

Axl receptor tyrosine kinase and its ligand Gas6 regulate several biological processes and are involved in both the onset and progression of tumor malignancies and autoimmune diseases. Based on its key role in these settings, Axl is considered a promising target for the development of molecules with therapeutic and diagnostic purposes. In this paper, we describe the molecular characterization of the recombinant Ig1 domain of Axl (Ig1 Axl) and its biochemical properties. For the first time, an exhaustive spectroscopic characterization of the recombinant protein through circular dichroism and fluorescence studies is also reported, as well as a binding analysis to its natural ligand Gas6, paving the way for the use of recombinant Ig1 Axl as a bait in drug discovery screening procedures aimed at the identification of novel and specific binders targeting the Axl receptor.