Abstract
Proline exists equilibria between endo/exo ring puckers and cis/trans peptide bond isomers. Prolyl cis/trans isomerization plays a critical role in protein folding and isomer-specific biochemical recognition. Since polyproline can form all-cis type I helices (PPI) or all-trans type II helices (PPII), it has been a valuable model to study the prolyl isomerization. Recent studies have shown that stereoelectronic effects influence the rate of PPII → PPI conversion and the fluoroproline substituted peptides have the most pronounced changes. In Chapter 2, we synthesized a series of host-guest peptides with 4-substituted fluoroproline incorporated into the N-terminus or the C-terminus and used a kinetic approach to explore terminal stereoelectronic effects on polyproline conformation. Time-dependent CD measurements revealed that incorporation of fluoroproline at the C-terminal end of polyproline has a large effect on PPII → PPI conversion, where a tri((2S,4R)-4-fluoroproline) sequence, (Flp)3, increases the transition barrier of PPII → PPI conversion by 1.53 kJ mol-1 while a tri((2S,4S)-4-fluoroproline) sequence, (flp)3, decreases the transition barrier by 4.61 kJ mol-1. In contrast, the same substitutions at the N-terminus only affect the transition barrier of PPII → PPI conversion by -0.03 to 0.10 kJ mol-1. Our results demonstrate stereoelectronic effects on PPII → PPI conversion are directional and provide an approach to establish the PPII → PPI transition mechanism. The conformational equilibria between ring puckers can be modulated by substitutions on the proline ring. Implanting an electron-withdrawing group on the 4S position of proline prefers a C-endo pucker and a cis peptide bond due to stereoelectronic effects, and favors polyproline I (PPI) helices rather than polyproline II (PPII) helices by preorganization. 4-Thiaproline (Thp) favors an endo ring pucker and a cis peptide bond, and we incorporated Thp into polyproline peptides to explore its effects on the peptide conformation in Chapter 3. We synthesized a series of peptides, including P5ThpP5, P11, Thp7, and P7, and characterized their structures by CD spectroscopy. The results show that Thp substitutions not only destabilize PPII helices but also decrease the tendency to form PPI helices. The density functional theory (DFT) analysis on Thp and Thp-containing oligopeptide reveals that the energy difference between exo and endo ring puckers is small for Thp, i.e. Thp only slightly favors the endo pucker. Such a small energy difference could lead to the coexistence of PPI and PPII in solution for Thp-containing peptides. Our data demonstrate that although Thp possesses a structure similar to proline, it has a significant impact on polyproline conformation. In a peptide or protein, the sequence with aromatic residues adjacent to proline residues shows a higher propensity in forming cis prolyl bonds due to aromatic-proline interactions or proline-aromatic interactions. The interactions are related to the electronegativity of aromatic- ring: an electron-rich aromatic ring relatively favored a cis amide bond. In Chapter 4, we incorporated aromatic amino acids (F, Y, W) into the N-terminal or the C-terminal end of polyproline to investigate aromatic-proline interactions on polyproline conformation and PPI ⟷ PPII interconversion kinetics. CD measurements reveal that the N-terminal aromatic-proline interaction significantly affects polyproline conformation more than the C-terminal aromatic-proline interaction. PPI stability is correlated with the strength of aromatic-proline interactions in the order of Y > W > F, while PPII stability is in an opposite order of F > W > Y. Time-dependent CD measurements reveal that aromatic-substitution effects are directional on PPII → PPI conversion but nondirectional on PPI → PPII conversion and the hydrophobicity of aromatic side chain may play a critical role in the conversion of PPI to PPII. Our data demonstrate that aromatic-proline interactions can modulate the stabilities of PPI and PPII conformations. Moreover, we proposed that aromatic-proline interactions may occur in the late stage of PPI folding process since the aromatic-proline interactions cannot speed the folding process.