Theoretical Conformational Analysis of Disulfide-Linked Tetrapeptides Ac-Cys-Pro-Xaa-Cys-NHMe Having Hydrophobic Xaa Amino-Acid Residues

Polymer Journal, Vol. 30, No. 3, pp 256---261 (1998) Theoretical Conformational Analysis of Disulfide-Linked Tetrapeptides Ac-Cys-Pro-Xaa-Cys-NHMe Having Hydrophobic Xaa Amino-Acid Residues Yuichirou ISHIKAWA, Yoshiaki HIRANO, Jun YOSHIMOTO,* Masahito OKA,**t and Toshia HAYASHI** Department of Applied Chemistry, Osaka Institute of Technology, Asahi-ku, Osaka 535, Japan * Graduate School of Human Informatics, Nagoya University, Chikusa-ku, Nagoya 464--01, Japan ** Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Osaka 593, Japan (Received September 18, 1997) ABSTRACT: Theoretical conformational analysis was carried out on four cyclic tetrapeptides Ac-Cys-Pro-XaaCys-NHMe (Xaa=Val, Phe, Leu, and norleucine) using Empirical Conformation Energy Program for Peptides (ECEPP) and optimization procedure for investigating the effects of differences in the hydrophonbic side-chain groups of Xaa residue on the {3-bend conformation at the Xaa-Pro portion of cyclic peptides having the disulfide linkage. Calculated results indicate that four cyclic Ac-Cys-Pro-Xaa-Cys-NHMe essentially form type III {3-bend at the Pro-Xaa portion, and also show fairly good agreement with experimental results of the NMR spectroscopy and X-ray crystallography for the tetrapeptides having Cys-Pro-Xaa-Cys sequence. KEY WORDS {3-Bend / Tetrapeptide / Disulfide Linkage / Molecular Mechanics / Empirical Conformation Energy Program for Peptides / For creating new artificial proteins, it is very important to design them through an a priori method based on the principle relations among three attributes of proteins, i.e., amino-acid sequences, conformations, and functions. From this viewpoint, we tried theoretical conformational analysis based on the molecular mechanics calculations to find all stable local minima in the whole conformational space of peptides 1- 3 and polypeptides, 4- 11 which are model molecules having key sequences in native proteins, and also showed that the lowest-energy conformations or the ensembles of the low-energy conformations of such molecules have reasonable structural characters which explain molecular functions of native proteins. Such conformational characters theoretically proposed for the peptides and polypeptides were also preliminary supported by experimental results. 12 - 15 Disulfide-linkages between two cystine residues are very important to introduce topological constraint into proteins, and contribute to stabilize the specific three-dimensional structure of proteins. In previous works, 16 - 18 theoretical conformational analysis was carried out on cyclic tetrapeptides Ac-Cys-Pro-GlyCys-NHMe16, Ac-Cys-Pro-Ala-Cys-NHMe 17 , and Ac-Cys-Pro-o-Ala-Cys-NHMe 18 using Empirical Conformation Energy Program for Peptides (ECEPP) 19 for designing amino-acid sequences for the loop portions of artificial functional proteins. Calculated results indicate that the disulfide-linkage stabilizes the specific P-bend structure at the Pro-Xaa portions. That is, cyclic AcCys-Pro-Gly-Cys-NHMe and Ac-Cys-Pro-o-AlaCys-NHMe form compactly folded conformations with type II P-bend at the Pro-Gly and Pro-o-Ala portions, respectively, and cyclic Ac-Cys-Pro-Ala-Cys-NHMe also forms those with type III P-bend at the Pro-Ala t To whom all correspondence should be addressed. 256 portion. It means that the bend type at the -CysPro-Xaa-Cys- sequence could be controlled by selecting the amino-acid residue Xaa (Xaa = Gly, Ala, and o-Ala). In this work, as a further step for investigating the effects of the difference in the side-chain groups on the conformational preference of the cyclic peptides with disulfide linkage, theoretical conformational analysis was carried out on four cyclic tetrapeptides Ac-CysPro-Xaa-Cys-NHMe (Xaa= Val, Phe, Leu, and norleucine abbreviated as Nie) using ECEPP 19 and optimization procedure. 20 THEORETICAL All conformational energy calculations were carried out on four disulfide-linked oligopeptides Ac-Cys-ProXaa-Cys-NHMe (Xaa= Val, Phe, Leu, and Nie) with the energy functions of ECEPP. 19 During minimizations using the Powell argorism, 20 all if; of Pro, (¢, if;, x 1, x2 • 1, x2·2) of Val,(¢, if;, x1, x2) of Phe, (¢, if;, X1, Xz, X3,1, X3,2) of Leu,(¢, 1/1, x1, X2, X3, X4) of Nie, and (¢, if;, x1) of cystine were allowed to vary. ¢ of Pro was fixed at - 75°. All other backbone dihedral angles were fixed at 180°. All combinations of single residue minima of Cys, Pro, and Xaa residues were used as starting conformations of minimization. Selected numbers of all stable single-residue minima were 21, 4, 10, 28, 15, and 60 for Cys, Pro, Val, Phe, Leu, and Nie, respectively. A bend (occurring at i + 1 and i + 2th residues) is defined as a conformation in which R 7 A (R is the distance between ith ca and i + 3th ca atoms) and is classified into one of the eleven types given in Table I of ref 21. A polar hydrogen atom and an oxygen or nitrogen atom with an interatomic distance of less than 2.3 A are regarded to be hydrogen-bonded. The conformational Conformation of Ac-Cys-Pro-Xaa--Cys-NHMe Table I. Conformational letter code DAAA DAAC EACE DFA*E EACD DFA*D A*AAA DAAA ECA*E ECA*E Minimum Energy Conformations• of Ac--Cys-Pro-Val-Cys-NHMe flEb Bend typed Ve kcalmol- 1 0.00 0.88 1.08 1.77 1.79 0.599 0.137 0.097 0.o31 0.030 III III I II I 1.93 2.12 2.20 2.29 2.50 0.024 0.017 0.015 0.013 0.009 II III III II II • All minima with llE <2.73 kcal mo1- 1 . Pro-Val and Val--Cys. b £0 III I III III <Pc,,1 1/tc,,1 -152 -152 -160 -152 -159 -152 60 -152 -157 -159 RESULTS AND DISCUSSION Stable Conformations of Ac-Cys-Pro-Val-Cys-NHMe There were 425 energy minima for Ac-Cys-ProVal-Cys-NHMe with AE<lO.0kcalmol- 1, and 10 of them (AE < 2. 73 kcal mol - 1) are shown in Table I. The lowest-energy conformation is a DAAA conformation (D, A, A and A are conformational letter codes for the Cysl, Pro, Val, and Cys4 residues, respectively.) taking type III-III double-bend at the Pro-Val-Cys portion as shown in Figure 1. This conformation shows excellently good agreement with the results of X-ray crystallography for the cyclic Ac-Cys-Pro-Val-Cys-NHMe by Falcomer et al. 26 That is, (</Jcysl• i/lcysl, ¢Pro, i/Jpro, </Jva1' i/lv.i, </Jcys4, i/lcys4• Xlys1, X2ys1, Xlys4, X2ys4, X••)=(-152, 89, -75, -18, -80, -24, -72, -47, -172, -139, -64, 77, 71) and (-135, 73, -60, -29, - 72, -18, - 73, -16, -170, -142, -66, 74, 78) for theoretically and experimentally evaluated values, respectively. An experimental value of 1 is not shown in Table V of ref 26. However, a stereo diagram of the crystal structure of the cyclic Ac-CysPro-Val-Cys-NHMe shown in Figure 6 of ref 26 indicates that the rotational state of the ca-C'1 bond of the Val residue is trans conformation. This is also consistent with the theoretically evaluated xi.1 = 175. Moreover, this conformation is stabilized by a hydrogen-bond, (Val)N · · · HN(Cys4) and a favorable hydrogen-bond-like interaction (...truncated)