Structural Stability of the Catalytic Domain of a Thermophilic Membrane Protein

ROMAN E; BREDESTON LM; ARGUELLO JM; GONZALEZ FLECHA FL

San Diego

Congreso; Biophysical Society 56th Annual Meeting; 2012

In a previous paper (J Mol Biol 297, 550–559, 2010) we have demonstrated that the thermophilic multidomain membrane protein CopA reversibly unfolds upon incubation with guanidinium hydrochloride (GndHCl), according to a two-state model. Because CopA is composed by several soluble and membrane domains, which has very different physicochemical properties, the following questions arise: is the two-state model a robust model to describe the unfolding of CopA?, where is encoded the thermophilicity of this protein? in this work, the catalytic domain of CopA (CopACD) was isolated and unfolded to understand its contribution to the whole protein stability. Our results showed that purified CopACD reversibly unfolds in the presence of GndHCl, as demonstrated by Trp fluorescence and circular dichroism, with DG, m, Cm and DCp values which are very different from that of CopA. Temperature denaturation of CopACD was also reversible and the Tm values were around 67
C in the absence of ligands, and around 70
C in the presence of Mg2þ/ATP. Both processes were well described by a two state model, however a GndHCl unfolding intermediate was detected by ANS fluorescence. We prepare two different singlecystein mutants in the nucleotide-binding (A520C) and phosphorylation (A660C) subdomains. These mutants were labelled with DTNB to obtain the adduct S-TNB, and the distance TNB-Trp upon GndHCl unfolding was evaluated by FRET. Preliminary results show no major differences in the unfolding of these subdomains. In summary, the obtained results showed that CopACD conserves the thermophilicity and thermostability of CopA, but unfolds in a more complex way. Then the apparent two-state behaviour of this multidomain protein would probably be the result of a combination of multiple undetected folding intermediates.