Synthesis and conformational analysis of cyclic cysine peptides

Abstract

Disulfide bridges are important determinants of protein and peptide structures. The formation of covalent crosslinks between cysteine residues that are far apart in the primary structure can result in the formation of loops and can restrict conformational flexibility. Disulfides can also play important functional roles in proteins involved in oxidation–reduction processes. Despite their well recognized significance, relatively few systematic attempts have been made to probe the conformational characteristics of cyclic peptide disulfides. Earlier investigations from this laboratory examined the stereochemistry of 14 membered disulfides containing Pro–X sequences as spacer segments (Venkatachalapathi, Y. V., Prasad, B. V. V., & Balaram, P. (1982) Biochemistry 21, 5502–5509; Ravi, A. (1983) Ph.D. Thesis, IISc, Bangalore; Ravi, A. & Balaram, P. (1984) Tetrahedron, 2577–2583). The main objective of the present investigation has been to characterize the conformational preferences of cyclic peptide disulfides ranging from 11 membered to 22 membered rings. Special attention has been given to 14 membered disulfides, which correspond to the active site sequences of the redox proteins thioredoxin and glutaredoxin. In all cases, model peptides were synthesized using classical solution phase procedures. Conformational analysis in solution was carried out primarily by NMR spectroscopy. The methods used include analysis of intramolecular hydrogen bonded NH groups and nuclear Overhauser effects (NOE) to probe spatial relationships between protons. This thesis is divided into nine chapters, outlined below. ________________________________________ Chapter I - Introduction Provides background on the parameters that determine peptide backbone and cystine side chain conformations. An account of spectroscopic techniques used to determine peptide backbone and disulfide conformations in solution is also presented. ________________________________________ Chapter II - Cyclic Biscystine Peptides Oxidation of the dithiol peptide Boc Cys(SH)–L Ala–Cys(SH)–NHCH yields: • an 11 membered cyclic peptide disulfide, • a 22 membered cyclic biscystine peptide. Studies on cyclic biscystine peptides of the type: Boc–Cys–X–Cys–NHCH | | S S | | NHCH –Cys–X–Cys–Boc (X = L Ala or D Ala) are described. These systems serve as excellent models for antiparallel sheet conformations. Solution conformations were established via NMR. A novel CD spectral pattern observed for these peptides is discussed. ________________________________________ Chapter III - 11 Membered Cyclic Peptide Disulfide Spectroscopic studies on Boc Cys–L Ala–Cys–NHCH show evidence for an “ideal turn” conformation involving both: • 3 1 (G ) • 1 3 (C ) hydrogen bonds in non polar solvents. In polar solvents, only a G turn appears to be retained. ________________________________________ Chapter IV - 17 Membered Cyclic Disulfides Two peptides were investigated: • Boc Cys–Ala–Aib–Gly–Cys–NHCH • Boc Cys–Gly–Leu–Gly–Cys–NHCH Results suggest an intramolecular antiparallel sheet structure with a turn at Aib(3)/Leu(3). Evidence of hydrogen bonding by several NH groups is presented. ________________________________________ Chapter V - 20 Membered Cyclic Peptide Disulfide Although oxytocin/vasopressin like disulfides have been widely studied in polar solvents, data in apolar solvents are scarce. The peptide: Boc Cys–Val–Aib–Ala–Leu–Cys–NHCH was synthesized and shown to adopt an antiparallel sheet conformation involving an Aib–Ala turn. Extensive NOE analysis further supports this structure. ________________________________________ Chapter VI - Synthesis of 14 Membered Redox Active Peptides The following model sequences mimicking active sites of thioredoxins and glutaredoxins were synthesized: 1. Boc Cys–Pro–Tyr–Cys–NHCH (Glutaredoxin models) 2. Boc Trp–Cys–Gly–Pro–Cys–NHCH (Thioredoxin models-E. coli, yeast, spinach, mammalian) 3. Boc Cys–Val–Tyr–Cys–NHCH (Phage T4 thioredoxin) 4. Boc Cys–Pro–Phe–Cys–NHCH (Model peptide for Tyr contribution study) ________________________________________ Chapter VII - Conformational Studies on 14 Membered Disulfides Key findings: • The thioredoxin mimicking peptide adopts consecutive turns, involving Gly–Pro and Pro–Cys sequences. • The Val–Tyr containing peptide supports a turn with Tyr at i+1. • Peptides with aromatic spacers (Pro–Tyr, Pro–Phe) show single conformers in CDCl but two species in (CD ) SO that exchange slowly on the NMR timescale. • An equilibrium between type I and type II turns is observed. Aromatic interactions at position 3 appear to increase the activation barrier for interconversion. ________________________________________ Chapter VIII - Fluorescence Studies and Redox Behaviour Presence of aromatic chromophores (Trp or Tyr) enables fluorescence monitoring of disulfide reduction. Rates of reduction by DTT were studied in methanol, water, and 8 M urea for thioredoxin and glutaredoxin model peptides. ________________________________________ Chapter IX - Summary and Conclusions Disulfide crosslinks effectively stabilize specific backbone conformations in small peptides. Studies on model redox active peptides show that conformational variability in 14 membered disulfide loops depends strongly on the identity of spacer residues.