Spectroscopic characterization of two soluble transducers from the archaeon Halobacterium salinarum

In the present study, structural aspects of the two soluble transducers, Ht rX and HtrXI, from the archaeon H. salinarum have been examined using UV ci rcular dichroism and steady-state fluorescence spectroscopies. Circular dic hroism (CD) data indicate that both HtrX and HtrXI exhibit salt-dependent p rotein folding. Under low-ionic-strength conditions (0.2 M NaCl or KCl) the CD spectra of HtrXI is similar to that of the Gdn-HCl- or urea-denatured f orms and is indicative of random coil structure. In contrast, the CD spectr um of HtrX under low-ionic-strength conditions contains roughly 85% alpha-h elical character, indicating a significant degree of folding. Addition of N aCl or KCl to solutions of HtrX or HtrXI results in CD features consistent with predominately alpha-helical character (>95%) for both proteins. In add ition, the transition points (i.e., ionic strengths at which the protein co nverts from random coil to alpha-helical character) are quite distinct and dependent upon the type of salt present (i.e., either NaCl or KCI). Accessi bility of tryptophan residues to the solvent was also examined for both Htr X and HtrXI in both folded and unfolded states using Kl quenching. The Ster n-Volmer constants obtained suggest that the tryptophans (Trp35 in HtrX and both Trp47 and Trp74 in HtrXI) are partially exposed to the solvent, indic ating that they are located near the surface of the protein in all three ca ses. Furthermore, fluorescence quenching with the single Trp mutants Trp74A Ia and Trp47AIa of HtrXI indicates different environments for these two res idues.