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A peptide recognition event leading to the native fold of E. coli thioredoxin: an NMR study Javier Santos 1, Andrés Binolfi2, Claudio Fernández2, and José M. Delfino1 1Departmento de Química Biológica e Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina. 2Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina. To whom correspondence should be addressed. E-mail: jsantos@qb.ffyb.uba.ar delfino@qb.ffyb.uba.ar The reduced form of fragment 1-93 of E. coli thioredoxin (TRX1-93) adopts in solution a partially folded conformation that consolidates into a native-like state upon interaction with peptide TRX94-108. A functional complex is formed spontaneously upon mixing both partners (Santos et al. 2007 Biochemistry 46, 5148-5159). Because isolated peptide TRX94-108 remains unstructured in solution and fragment TRX1-93 exhibits features of a molten globule state, folding of both peptide and fragment should occur concomitantly. We have recently shown that specific interactions involving L99, F102 and L103 between peptide TRX94-108 and partially folded TRX1-93 play a key role in stabilizing the native-like tertiary structure of the complex (Santos et al. 2009 Biochemistry 48, 595-607). In this context, we hypothesize that folding depends on the formation of a cooperative tertiary unit based on the interaction between a-helix 5 of peptide TRX94-108 and a-helix 3 of fragment TRX1-93. This view is supported by results from molecular dynamics simulations. Here we report an NMR-based approach aimed at uncovering the molecular events leading to complex formation.