proteins STRUCTURE O FUNCTION O BIOINFORMATICS Gain of local structure in an amphipathic peptide does not require a specific tertiary framework Ernesto A. Roman, 1 Pablo Rosi, 1 Mariano C. Gonza ´lez Lebrero, 1 Rodolfo Wuilloud, 2 F. Luis Gonza ´lez Flecha, 1 Jose ´ M. Delfino, 1 and Javier Santos 1,3 * 1 Department of Biological Chemistry and Institute of Biochemistry and Biophysics (IQUIFIB), School of Pharmacy and Biochemistry, University of Buenos Aires, Junı ´n 956, C1113AAD, Buenos Aires, Argentina 2 Laboratory of Environmental Research and Services of Mendoza (LISAMEN), CCT-CONICET Mendoza. Av. Ruiz Leal S/N Parque General San Martı´n, M 5502 IRA Mendoza, Argentina 3 Department of Science and Technology, University of Quilmes, Roque Sa ´enz Pen ˜a 352, B1876XD, Bernal, Argentina INTRODUCTION A glance at the sequence of a protein does not directly reveal the existence of structure. However, it encrypts the physico- chemical properties needed to assemble itself into a three- dimensional arrangement. 1 A straightforward example that illustrates how a sequence determines a structure is the case of natural amphipathic a-helices. The amphipathicity of an a-he- lix is a property that becomes manifest in the folded conforma- tion. Amphipathic a-helices occur ubiquitously in globular pro- teins where they simultaneously interact on one face with water molecules and on the opposite face with other residues of the hydrophobic core. In general, local (near in sequence) and terti- ary interactions contribute together to attain structure. Never- theless, even for this deceivingly simple case, we do not know about the relative weight and complex relationship between these two classes of interactions to provide stability to this struc- tural module. For example, the grafting of nonhomologous am- phipathic a-helices into b-lactamase results in native-like states highlighting the robustness of the folding process. 2 On the other hand, despite the presence of stabilized individual helices, defects in the acquisition of native-like packing were observed for a de novo designed triple-helix bundle. 3 Furthermore, pro- tein structure seems to balance both thermodynamic stability and conformational specificity toward the folded state over the ensemble of unfolded/partially folded conformations. An excel- lent example is the family of a-2 dimeric four-helix bundle, and Additional supporting information may be found in the online version of this article. Grant sponsors: Agencia Nacional de Promocio ´ n Cientı ´fica y Tecnolo ´ gica (ANPCyT), Consejo Nacional de Investigaciones Cientı´ficas y Te ´cnicas (CONICET), Universidad de Buenos Aires (UBACyT), Universidad Nacional de Quilmes (UNQ); Grant sponsor: ANPCyT; Grant number: PME2003-0026. Ernesto A. Roman and Pablo Rosi contributed equally to this work. *Correspondence to: Javier Santos, Department of Biological Chemistry and Institute of Biochemistry and Biophysics (IQUIFIB), School of Pharmacy and Biochemistry, University of Buenos Aires, Junı´n 956, C1113AAD, Buenos Aires, Argentina. E-mail: jsantos@qb.ffyb.uba.ar. Received 11 February 2010; Revised 18 May 2010; Accepted 20 May 2010 Published online 8 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.22789 ABSTRACT In this work, we studied how an amphipathic peptide of the surface of the globular protein thioredoxin, TRX94- 108, acquires a native-like structure when it becomes involved in an apolar interaction network. We designed peptide variants where the tendency to form a-helical conformation is modulated by replacing each of the leu- cine amino acid residues by an alanine. The induction of structure caused by sodium dodecyl sulfate (SDS) bind- ing was studied by capillary zone electrophoresis, circu- lar dichroism, DOSY-NMR, and molecular dynamics sim- ulations (MDS). In addition, we analyzed the strength of the interaction between a C18 RP-HPLC matrix and the peptides. The results presented here reveal that (a) criti- cal elements in the sequence of the wild-type peptide sta- bilize a SDS/peptide supramolecular cluster; (b) the hydrophobic nature of the interaction between SDS mol- ecules and the peptide constrains the ensemble of confor- mations; (c) nonspecific apolar surfaces are sufficient to stabilize peptide secondary structure. Remarkably, MDS shed light on a contact network formed by a limited number of SDS molecules that serves as a structural scaf- fold preserving the helical conformation of this module. This mechanism might prevail when a peptide with low helical propensity is involved in structure consolidation. We suggest that folding of peptides sharing this feature does not require a preformed tightly-packed protein core. Thus, the formation of specific tertiary interactions would be the consequence of peptide folding and not its cause. In this scenario, folding might be thought of as a process that includes unspecific rounds of structure sta- bilization guiding the protein to the native state. Proteins 2010; 78:2757–2768. V V C 2010 Wiley-Liss, Inc. Key words: thioredoxin; SDS; helical propensity; leucine pair of interactions; molecular dynamics simulations; secondary structure stability; folding. V V C 2010 WILEY-LISS, INC. PROTEINS 2757