Structural and sequence features of beta-turns in beta-hairpins Bharat Madan, and Sun-Gu Lee* Department of Chemical and Biochemical Engineering, Pusan National University, Busan 609-735, Republic of Korea TEL: +82-51-583-8343, FAX: +82-51-512-8563 Abstract Beta-turns in beta-hairpins have been implicated as important sites in protein folding. In particular, two residue β-turns, the most abundant connecting elements in beta-hairpins, have been a major target for engineering protein stability and folding. In this study, we attempted to investigate and update the structural and sequence properties of two residue turns in beta-hairpins with a large data set. For this, 3977 beta-turns were extracted from 2394 non-homologous protein chains and analyzed. First, the distribution, dihedral angles and twists of two residue turn types were determined, and compared with previous data. The trend of turn type occurrence and most structural features of the turn types were similar to previous results, but for the first time Type II turns in beta-hairpins were identified. Second, sequence motifs for the turn types were devised based on amino acid positional potentials of two-residue turns, and their distributions were examined. From this study, we could identify code-like sequence motifs for the two residue beta-turn types. Finally, structural and sequence properties of beta-strands in the beta-hairpins were analyzed, which revealed that the beta-strands showed no specific sequence and structural patterns for turn types. The analytical results in this study are expected to be a reference in the engineering or design of beta-hairpin turn structures and sequences. Background Beta-turns account for nearly 25% of the protein architecture. They are found at regions of chain reversal in proteins and are an integral part of beta-hairpin. They consists of 4 residues (i to i+3) and are characterized into various types based on phi/psi angles of residues at i+1 and i+2 positions. Four main types commonly found in proteins are types – I, II, I’ and II’. Figure 1. Four most common types of beta-turns found in proteins Objective Why beta-hairpin beta-turns ? PDB (Protein Data Bank) Structural and sequence characterization of two residue β-hairpin turns Two beta-strands + turn = beta-hairpin. Turns are an integral part of beta-hairpins. Turns help in bringing the two strands of a hairpin farther in primary sequence, closer in 3-D structure. Thus, help in folding of a beta-hairpin. Therefore, turns can act as nucleation sites of protein folding. Amino acid code identification for turns Turn Type distribution Positional Preferences of amino-acids in turns Dihedral angle distribution Sheet-twist distribution Beta-turn twist distribution Extract structures containing two-residue beta-hairpins Figure 2. Overview of the entire study. Results Turn type distribution in beta-hairpins Turn twist distribution of beta-turns Sheet twist distribution Figure 3.. Turn type distribution in β-hairpins. Dihedral angle preference of beta-turns Position i+1 Type I’ Position i+2 Figure 5. Turn twist distribution of beta-turns. Amino acid preferences in beta-turns Type-I’ Type-II’ Type II’ Figure 7.Sheet twist distribution of β-hairpins. Conclusion Types I’ and II’ beta-turns are found predominantly in β- hairpins. Type I’ is evolutionarily more conserved than the other turn types. Each turn type has distinct residue preference. This suggests that turn sequences do not occur randomly in nature. The higher occurrence of type I’ turns , with defined residue preference suggests they could be efficient in protein folding. The results presented in this study can be used in the efficient design of beta-hairpin β-turns. Type-I Type-II Type I Figure 6.Positional potentials of amino acids in beta-turns. Amino acid codes for beta-turns References Type II Based on the most preferred amino acids at positions i+1 and i+2, amino acid codes were defined for each beta-turn type . 33. Chou PY, Fasman GD. β-turns in proteins. J Mol Biol 1977;115:135-175. 28. Gunasekaran K, Ramakrishnan C, Balaram P. Beta-hairpins in proteins revisited: lessons for de novo design. Protein Eng 1997;10:1131-1141. 25. de Alba E, Rico M, Jimenez MA. The turn sequence directs beta-strand alignment in designed beta-hairpins. Protein Sci 1999;8:2234-2244. 8. Hutchinson EG, Thornton JM. A revised set of potentials for beta-turn formation in proteins. 1994;3:2207-2216. Madan B, Seo SY, Lee SG. Structural and sequence features of two residue turns in beta-hairpins. Proteins 2014. ( doi/10.1002/prot.24526) Table 1. Amino acid codes for beta-turns Type I’ Type II’ Type I Type II X-XP,G-G-XG X-G-XG-XG X-XP,G-XG-G X-P-XG-G X-P-G-XG X= all 20 amino acids except the superscripted ones. Figure 4. Ramachandran plots of Φ/Ψ preference for beta-turn residues i+1 and i+2.