Showing papers by "Padmanabhan Balaram published in 2006"

NMR Analysis of Aromatic Interactions in Designed Peptide \beta -Hairpins

Abstract: Designed octapeptide \beta -hairpins containing a central $ ^DPro-Gly$ segment have been used as a scaffold to place the aromatic residues Tyr and Trp at various positions on the antiparallel \beta -strands. Using a set of five peptide hairpins, aromatic interactions have been probed across antiparallel \beta-sheets, in the non-hydrogen bonding position $(Ac-L-Y-V-^DP-G-L-Y/W-V-OMe: peptides 1 and 2)$, diagonally across the strands $(Boc-Y/W-L-V-^DP-G-W-L-V-OMe: peptides 3 and 6)$, and along the strands at positions iand i+ 2 $(Boc-L-L-V-^DP-G-Y-L-W-OMe: peptide 4)$. Two peptides served as controls $(Boc-L-L-V-^DP-G-Y-W-V-OMe: peptide 5; Boc-L-Y-V-^DP-G-L-L-V-OMe: peptide 7)$ for aromatic interactions. All studies have been carried out using solution NMR methods in $CDCI_3 + 10% DMSO- d_6$ and have been additionally examined in $CD_3OH$ for peptides 1 and 2. Inter-ring proton-proton nuclear Overhauser effects (NOEs) and upfield shifted aromatic proton resonances have provided firm evidence for specific aromatic interactions. Calculated NMR structures for peptides 1 and 2, containing aromatic pairs at facing non-hydrogen bonded positions, revealed that T-shaped arrangements of the interacting pairs of rings are favored, with ring current effects leading to extremely upfield chemical shifts and temperature dependences for specific aromatic protons. Anomalous far-UV CD spectra appeared to be a characteristic feature in peptides where the two aromatic residues are spatially proximal. The observation of the close approach of aromatic rings in organic solvents suggests that interactions of an electrostatic nature may be favored. This situation may be compared to the case of aqueous solutions, where clustering of aromatic residues is driven by solvophobic (hydrophobic) forces.

Design of a peptide hairpin containing a central three-residue loop.

Abstract: The construction of a designed $\beta$-hairpin structure, containing a central three-residue loop has been successfully achieved in the synthetic nonapeptide $Boc-Leu-Phe-Val-^DPro-^LPro-^DAla-Leu-Phe-Val-OMe$ (2). The design is based on expanding the two-residue loop established in the peptide $\beta$-hairpin $Boc-Leu-Phe-Val-^DPro-^LPro-Leu-Phe-Val-OMe$ (1). Characterization of the registered $\beta$-hairpins in peptides 1 and 2 is based on the observation of key nuclear Overhauser effects (NOEs) in $CDCl_3$ and $CD_3OH$. Solvent titration and temperature dependence of NH chemical shifts establish the identity of NH groups involved in interstrand hydrogen bonding. In peptide 2, the antiparallel registry is maintained, with the formation of a $^DPro-^LPro-^DAla$ loop, stabilized by a $5\rightarrow1$ hydrogen bond between Val3 CO and Leu7 NH groups $(C_{13}, \alpha-turn)$ and a $3\rightarrow1$ hydrogen bond between $^DPro4$ CO and $^DAla6$ NH groups $(C_7, \gamma-turn)$. NMR derived structures suggest that in peptide 2, $^DAla(6)$ adopts an $\alpha_L$ conformation. In peptide 1, the $^DPro-^LPro$ segment adopts a type II' $\beta$-turn. Replacement of $^DAla (6)$ in peptide 2 by $^LAla$ in peptide 3 yields a $\beta$-hairpin conformation, with a central $^DPro-^LPro$ two-residue loop. Strand slippage at the C-terminus results in altered registry of the antiparallel strands.

Diproline templates as folding nuclei in designed peptides. Conformational analysis of synthetic peptide helices containing amino terminal Pro-Pro segments.

Abstract: The effect of N-terminal diproline segments in nucleating helical folding in designed peptides has been studied in two model sequences Piv-Pro-Pro-Aib-Leu-Aib-Phe-OMe (1) and Boc-Aib-Pro-Pro-Aib-Val-Ala-Phe-OMe (2). The structure of 1 in crystals, determined by X-ray diffraction, reveals a helical (RR) conformation for the segment residues 2 to 5, stabilized by one 4 -> 1 hydrogen bond and two 5 -> 1 interactions. The N-terminus residue, Pro(1) adopts a polyproline II (P-II) conformation. NMR studies in three different solvent systems support a conformation similar to that observed in crystals. In the apolar solvent CDCl3, NOE data favor the population of both completely helical and partially unfolded structures. In the former, the Pro-Pro segment adopts an alpha(R)-alpha(R) conformation, whereas in the latter, a P-II-alpha(R) structure is established. The conformational equilibrium shifts in favor of the P-II-alpha(R) structure in solvents like methanol and DMSO. A significant population of the Pro(1)- Pro(2) cis conformer is also observed. The NMR results are consistent with the population of at least three conformational states about Pro- Pro segment: trans alpha(R)-alpha(R), trans P-II-alpha(R) and cis P-II-alpha(R). Of these, the two trans conformers are in rapid dynamic exchange on the NMR time scale, whereas the interconversion between cis and trans form is slow. Similar results are obtained with peptide 2. Analysis of 462 diproline segments in protein crystal structures reveals 25 examples of the alpha(R)-alpha(R) conformation followed by a helix. Modeling and energy minimization studies suggest that both P-II-alpha(R) and alpha(R)-alpha(R) conformations have very similar energies in the model hexapeptide 1

De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by Fourier transform ion cyclotron resonance mass spectrometry.

Abstract: T-1-family conotoxins belong to the T-superfamily and are composed of 10-17 amino acids. They share a common cysteine framework and disulfide connectivity and exhibit unusual posttranslational modifications, such as tryptophan bromination, glutamic acid carboxylation, and threonine glycosylation. We have isolated and characterized a novel peptide, Mo1274, containing 11 amino acids, that shows the same cysteine pattern, -CC-CC, and disulfide linkage as those of the T-1-family members. The complete sequence, GNWCCSARVCC, in which W denotes bromotryptophan, was derived from MS-based de novo sequencing. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), infrared multiphoton dissociation, and collision-induced dissociation served to detect and localize the tryptophan bromination. The bromine contributes a distinctive isotopic distribution in all fragments that contain bromotryptophan. ECD fragmentation results in the loss of bromine and return to the normal isotopic distribution. Disulfide connectivity of Mo1274, between cysteine pairs 1-3 and 2-4, was determined by mass spectrometry in combination with chemical derivatization employing tris(2-carboxyethyl)phosphine, followed by differential alkylation with N-ethylmaleimide and iodoacetamide. The ECD spectra of the native and partially modified peptide reveal a loss of bromine in a process that requires the presence of a disulfide bond.

Tandem electrospray mass spectrometric studies of proton and sodium ion adducts of neutral peptides with modified N- and C-termini: Synthetic model peptides and microheterogeneous peptaibol antibiotics

Abstract: The fragmentations of $[M+H]^+$ and $[M+Na]^+$ adducts of neutral peptides with blocked N- and C-termini have been investigated using electrospray ion trap mass spectrometry. The N-termini of these synthetically designed peptides are blocked with a tertiarybutyloxycarbonyl (Boc) group, and the C-termini are esterified. These peptides do not possess side chains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage patterns of the protonated peptides are strikingly different from those of sodium ion adducts. While the loss of the N-terminal blocking group occurs quite readily in the case of MS/MS of $[M+Na]^+$, the cleavage of the C-terminal methoxy group seems to be a facile process in the case of MS/MS of $[M+H]^+$. Fragmentation of the protonated adducts yields only $b_n$ ions, while $y_n$ and $a_n$ ions are predominantly formed from the fragmentation of sodium ion adducts. The $a_n$ ions arising from the fragmentation of $[M+Na]^+$ lack the N terminal Boc group (and are here termed $a_n$ * ions). MS/MS of $[M+Na]^+$ species also yields $b_n$ ions of substantially lower intensities that lack the N-terminal Boc group ($b_n$*). A similar distinction between the fragmentation patterns of proton and sodium ion adducts is observed in the case of peptides possessing an N-terminal acetyl group. An example of the fragmentation of the $H^+$ and $Na^+$ adducts of a naturally occurring peptaibol from a Trichoderma species confirms that fragmentation of these two ionized species yields complementary information, useful in sequencing natural peptides. Inspection of the isotopic pattern of $b_n$ ions derived from $[M+H]^+$ adducts of peptaibols provided insights into the sequences of microheterogeneous samples. This study reveals that the combined use of protonated and sodium ion adducts should prove useful in de novo sequencing of peptides, particularly of naturally occurring neutral peptides with modified N- and C-termini, for example, peptaibols.

Hybrid Peptide Hairpins Containing α- and ω-Amino Acids: Conformational Analysis of Decapeptides with Unsubstituted β-, γ-, and δ-Residues at Positions 3 and 8

Abstract: The effects of inserting unsubstituted -amino acids into the strand segments of model -hairpin peptides was investigated by using four synthetic decapeptides, Boc-Leu-Val-Xxx-Val-D-Pro-Gly-Leu-Xxx-Val-Val-OMe: peptide 1 (Xxx=Gly), peptide 2 (Xxx=$\beta$Gly=$\beta$ahGly=homoglycine, -$\beta$glycine), peptide 3 (Xxx=$\gamma$Abu=-$\gamma$aminobutyric acid), peptide 4 (Xxx=$\delta$Ava=-$\delta$aminovaleric acid). $^1H\hspace{2mm}NMR$ studies (500 MHz, methanol) reveal several critical cross-strand NOEs, providing evidence for -$\beta$hairpin conformations in peptides 2-4. In peptide 3, the NMR results support the formation of the nucleating turn, however, evidence for cross-strand registry is not detected. Single-crystal X-ray diffraction studies of peptide 3 reveal a -$\beta$hairpin conformation for both molecules in the crystallographic asymmetric unit, stabilized by four cross-strand hydrogen bonds, with the $\gamma$Abu residues accommodated within the strands. The D-Pro-Gly segment in both molecules (A,B) adopts a type II -$\beta$turn conformation. The circular dichroism spectrum for peptide 3 is characterized by a negative CD band at 229 nm, whereas for peptides 2 and 4, the negative band is centered at 225 nm, suggesting a correlation between the orientation of the amide units in the strand segments and the observed CD pattern.

Non-protein amino acids in the design of secondary structure scaffolds.

Abstract: The use of stereochemically constrained amino acids permits the design of short peptides as models for protein secondary structures. Amino acid residues that are restrained to a limited range of backbone torsion angles (phi-psi) may be used as folding nuclei in the design of helices and beta-hairpins. alpha-Amino-isobutyric acid (Aib) and related Calphaalpha dialkylated residues are strong promoters of helix formation, as exemplified by a large body of experimentally determined structures of helical peptides. DPro-Xxx sequences strongly favor type II' turn conformations, which serve to nucleate registered beta-hairpin formation. Appropriately positioned DPro-Xxx segments may be used to nucleate the formation of multistranded antiparallel beta-sheet structures. Mixed (alpha/beta) secondary structures can be generated by linking rigid modules of helices and beta-hairpins. The approach of using stereochemically constrained residues promotes folding by limiting the local structural space at specific residues. Several aspects of secondary structure design are outlined in this chapter, along with commonly used methods of spectroscopic characterization.

Structural studies of model peptides containing beta-, gamma- and delta-amino acids.

Abstract: The crystal structures of five model peptides Piv-Pro-Gly-NHMe (1), Piv-Pro-βGly-NHMe (2), Piv-Pro-βGly-OMe (3), Piv-Pro-δAva-OMe (4) and Boc-Pro-γAbu-OH (5) are described (Piv: pivaloyl; NHMe: N-methylamide; βGly: β-glycine; OMe: O-methyl ester; δAva: δ-aminovaleric acid; γAbu: γ-aminobutyric acid). A comparison of the structures of peptides 1 and 2 illustrates the dramatic consequences upon backbone homologation in short sequences. 1 adopts a type II β-turn conformation in the solid state, while in 2, the molecule adopts an open conformation with the β-residue being fully extended. Piv-Pro-βGly-OMe (3), which differs from 2 by replacement of the C-terminal NH group by an O-atom, adopts an almost identical molecular conformation and packing arrangement in the solid state. In peptide 4, the observed conformation resembles that determined for 2 and 3, with the δAva residue being fully extended. In peptide 5, the molecule undergoes a chain reversal, revealing a β-turn mimetic structure stabilized by a C–H⋯O hydrogen bond.

Aggregation modes in sheets formed by protected β-amino acids and β-peptides

Abstract: The crystal structures of four protected β-amino acid residues, Boc-(S)-β3-HAla-NHMe (1); Boc-(R)-β3-HVal-NHMe (2); Boc-(S)-β3-HPhe-NHMe (3); Boc-(S)-β3-HPro-OH (6) and two β-dipeptides, Boc-(R)-β3-HVal-(R)-β3-HVal-OMe (4); Boc-(R)-β3-HVal-(S)-β3-HVal-OMe (5) have been determined. Gauche conformations about the Cβ–Cα bonds (θ ∼ ±60°) are observed for the β3-HPhe residues in 3 and all four β3-HVal residues in the dipeptides 4 and 5. Trans conformations (θ ∼ 180°) are observed for β3-HAla residues in both independent molecules in 1 and for the β3-HVal and β3-HPro residues in 2 and 6, respectively. In the cases of compounds 1–5, molecules associate in the crystals via intermolecular backbone hydrogen bonds leading to the formation of sheets. The polar strands formed by β3-residues aggregate in both parallel (1, 3, 5) and antiparallel (2, 4) fashion. Sheet formation accommodates both the trans and gauche conformations about the Cβ–Cα bonds.

Structure formation in short designed peptides probed by proteolytic cleavage.

Abstract: The formation of local structure, in short peptides has been probed by examining cleavage patterns and rates of proteolysis of designed sequences with a high tendency to form β-hairpin structures. Three model sequences which bear fluorescence donor and acceptor groups have been investigated: Dab-Gaba-Lys-Pro-Leu-Gly-Lys-Val-Xxx-Yyy-Glu-Val-Ala-Ala-Cys-Lys-NH2 i EDANS Xxx-Yyy: Peptide 1=DPro-LPro, Peptide 2=DPro-Gly, Peptide 3=Leu-Ala Fluorescence resonance energy transfer (FRET) provides a convenient probe for peptide cleavage. MALDI mass spectrometry has been used to probe sites of cleavage and CD spectroscopy to access the overall backbone conformation using analog sequences, which lack strongly absorbing donor and acceptor groups. The proteases trypsin, subtilisin, collagenase, elastase, proteinase K and thermolysin were used for proteolysis and the rates of cleavage determined. Peptide 3 is the most susceptible to cleavage by all the enzymes except thermolysin, which cleaves all three peptides at comparable rates. Peptides 1 and 2 are completely resistant to the action of trypsin, suggesting that β-turn formation acts as a deterrent to proteolytic cleavage.

Structural studies of model peptides \beta-, \gamma- containing and \delta- amino acids

Abstract: The crystal structures of five model peptides Piv-Pro-Gly-NHMe (1), Piv-Pro-\beta Gly-NHMe (2), Piv-Pro- \beta Gly-OMe (3), Piv-Pro- \delta Ava-OMe (4) and Boc-Pro- \gamma Abu-OH (5) are described (Piv: pivaloyl; NHMe: N-methylamide; \beta Gly: \beta-glycine; OMe: O-methyl ester; \delta Ava: \delta -aminovaleric acid; \gamma Abu: \gamma -aminobutyric acid). A comparison of the structures of peptides 1 and 2 illustrates the dramatic consequences upon backbone homologation in short sequences. 1 adopts a type II \beta -turn conformation in the solid state, while in 2, the molecule adopts an open conformation with the \beta -residue being fully extended. Piv-Pro- \beta Gly-OMe (3), which differs from 2 by replacement of the C-terminal NH group by an O-atom, adopts an almost identical molecular conformation and packing arrangement in the solid state. In peptide 4, the observed conformation resembles that determined for 2 and 3, with the \delta Ava residue being fully extended. In peptide 5, the molecule undergoes a chain reversal, revealing a \beta -turn mimetic structure stabilized by a $C-H...O$ hydrogen bond.