Stability and Folding of a TRX fragment.

JAVIER SANTOS; MAURICIO P. SICA; CRISTINA MARINO BUSLJE; MARIO R. ERMÁCORA; JOSÉ MARÍA DELFINO

Long Beach, California, USA.

Congreso; Biophysica Society meeting; 2008

IUPAB

  Molecular recognition between C-terminal helix 5 and fragment TRX1-93 of E. coli thioredoxin (TRX) embodies reciprocal structure selection where both partners gain order. Packing of helix 5 against the remainder of the protein stabilizes the TRX fold (Santos et al. 2007 Biochemistry 46, 5148-5159), pointing to the potential role on specific stabilization played by a row of four L residues (LSKGQLKEFLDANLA) clustered along one side of the helix. The significance of contacts involving L99, E101, F102, L103 and L107 to stabilize secondary structure was assessed by alanine mutagenesis. The helix-forming trend of peptide variants was measured by the strength of TFE-induced far-UV CD bands. Binding of peptide variants of TRX94-108 to the reduced form of fragment TRX1-93 was explored by near-UV CD, MS and MD simulations. The last five residues of helix 5 in M. tuberculosis TRX insert into a groove adjacent to the active site (Hall et al. 2006 Acta Crystallogr D Biol Crystallogr 62, 1453-1457). Here, the C-terminal helix is somewhat relaxed and the particular crystal packing might help stabilize this unusual conformation. Thus, native TRX conformation would persist even if helix 5 becomes disorganized. In addition, it is a fact that oxidized full-length E. coli TRX is stabilized by ~3.4 kcal/mol as compared to the reduced form. In this vein, we demonstrated that -upon C32-C35 disulfide bridge formation- >50% of fragment TRX1-93 acquires a native-like state, as evidenced by near-UV CD and MS. In the view of energy landscapes, reduced TRX1-93 would map a rather flat funnel where the molten globule conformation is populated. By contrast, the energy surface would deepen (or become smoother) when a specific peptide recognition event or an ‘allosteric’ covalent backbone stiffening interaction contribute to the consolidation of the native form.