Showing papers by "Padmanabhan Balaram published in 2012"

Distinct Disulfide Isomers of μ-Conotoxins KIIIA and KIIIB Block Voltage-Gated Sodium Channels

Abstract: In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of mu-conotoxin KIIIA, which was predicted originally to have a C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related mu-conotoxins. The two major isomers of synthetic mu-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of Na(v)1.2 (K-d values of 5 and 230 nM, respectively). The solution structure for mu-KIIIA based on nuclear magnetic resonance data was recalculated with the C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the mu-KIIIA structure calculated with the incorrect C1-C9,C2-C15,C4-C16] disulfide pattern, with an alpha-helix spanning residues 7-12. In addition, the major folding isomers of mu-KIIIB, an N-terminally extended isoform of mu-KIIIA, identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as mu-KIIIA, and both blocked Na(v)1.2 (K-d values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic mu-KIIIA and mu-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of mu-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail's repertoire of active peptides.

Aromatic interactions in model peptide β-hairpins: ring current effects on proton chemical shifts.

Abstract: Crystal structures of eight peptide β-hairpins in the sequence Boc-Leu-Phe-Val-Xxx-Yyy-Leu-Phe-Val-OMe revealed that the Phe(2) and Phe(7) aromatic rings are in close spacial proximity, with the centroid–centroid distance (Rcen) of 4.4–5.4 A between the two phenyl rings. Proton NMR spectra in chloroform and methanol solution reveal a significant upfield shift of the Phe(7) Cδ,δ′H2 protons (6.65–7.04 ppm). Specific assignments of the aromatic protons have been carried out in the peptide Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe (6). The anticipated ring current shifts have been estimated from the aromatic ring geometrics observed in crystals for all eight peptides. Only one of the Cδ,δ′H proton lies in the shielding zone with rapid ring flipping, resulting in averaging between the two extreme chemical shifts. An approximate estimate of the population of conformations, which resemble crystal state orientation, may be obtained. Key nuclear Overhauser effects (NOEs) between facing Phe side chains provide support for close similarity between the solid state and solution conformation. Temperature dependence of aromatic ring proton chemical shift and line widths for peptide 6 (Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe) and the control peptide Boc-Leu-Val-Val-DPro-Gly-Leu-Phe-Val-OMe establish an enhanced barrier to ring flipping when the two Phe rings are in proximity. Modeling studies suggest that small, conformational adjustment about CαCβ (χ1) and CβCγ (χ2) bonds of both the Phe residues may be required in order to permit unhindered, uncorrelated flipping of both the Phe rings. The maintenance of the specific aromatic ring orientation in organic solvents provides evidence for significant stabilizing interaction. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 185–194, 2012.

Structural Characterization of Backbone-Expanded Helices in Hybrid Peptides: (αγ)n and (αβ)n Sequences with Unconstrained β and γ Homologues of L-Val

Abstract: Learning your I±I²I³'s: The diversity of hydrogen-bonding patterns in backbone-expanded hybrid helices is shown by crystal-structure determination of several oligomeric peptides (see scheme; C=gray; H=white; O=red; N=blue). C 12 helices were observed in the I±I³ peptide series for n=2-8. In comparison, the I±I± peptide and I±I² peptide sequences show C 10 and mixed C 14/C 15 helices, respectively. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Asparagine and glutamine differ in their propensities to form specific side chain-backbone hydrogen bonded motifs in proteins.

Abstract: Short range side chain-backbone hydrogen bonded motifs involving Asn and Gln residues have been identified from a data set of 1370 protein crystal structures (resolution ≤ 1.5 A). Hydrogen bonds involving residues i - 5 to i + 5 have been considered. Out of 12,901 Asn residues, 3403 residues (26.4%) participate in such interactions, while out of 10,934 Gln residues, 1780 Gln residues (16.3%) are involved in these motifs. Hydrogen bonded ring sizes (C(n), where n is the number of atoms involved), directionality and internal torsion angles are used to classify motifs. The occurrence of the various motifs in the contexts of protein structure is illustrated. Distinct differences are established between the nature of motifs formed by Asn and Gln residues. For Asn, the most highly populated motifs are the C(10)(CO(δ)(i) …NH(i + 2)), C(13)(CO(δ)(i) …NH(i + 3)) and C(17)(N(δ)H(i) …CO(i - 4)) structures. In contrast, Gln predominantly forms C(16)(CO(e)(i) …NH(i - 3)), C(12)(N(e)H(i) …CO(i - 2)), C(15)(N(e)H(i) …CO(i - 3)) and C(18)(N(e)H(i) …CO(i - 4)) motifs, with only the C(18) motif being analogous to the Asn C(17) structure. Specific conformational types are established for the Asn containing motifs, which mimic backbone β-turns and α-turns. Histidine residues are shown to serve as a mimic for Asn residues in side chain-backbone hydrogen bonded ring motifs. Illustrative examples from protein structures are considered.

β-turn analogues in model αβ-hybrid peptides: Structural characterization of peptides containing β 2,2Ac 6c and β 3,3Ac 6c residues

Abstract: The effect of gem-dialkyl substituents on the backbone conformations of β-amino acid residues in peptides has been investigated by using four model peptides: Boc-Xxx-β(2,2)Ac(6)c(1-aminomethylcyclohexanecarboxylic acid)-NHMe (Xxx = Leu (1), Phe (2); Boc = tert-butyloxycarbonyl) and Boc-Xxx-β(3,3)Ac(6)c(1-aminocyclohexaneacetic acid)-NHMe (Xxx = Leu (3), Phe (4)). Tetrasubstituted carbon atoms restrict the ranges of stereochemically allowed conformations about flanking single bonds. The crystal structure of Boc-Leu-β(2,2)Ac(6)c-NHMe (1) established a C(11) hydrogen-bonded turn in the αβ-hybrid sequence. The observed torsion angles (α(ϕ≈-60°, ψ≈-30°), β(ϕ≈-90°, θ≈60°, ψ≈-90°)) corresponded to a C(11) helical turn, which was a backbone-expanded analogue of the type III β turn in αα sequences. The crystal structure of the peptide Boc-Phe-β(3,3)Ac(6)c-NHMe (4) established a C(11) hydrogen-bonded turn with distinctly different backbone torsion angles (α(ϕ≈-60°, ψ≈120°), β(ϕ≈60°, θ≈60°, ψ≈-60°)), which corresponded to a backbone-expanded analogue of the type II β turn observed in αα sequences. In peptide 4, the two molecules in the asymmetric unit adopted backbone torsion angles of opposite signs. In one of the molecules, the Phe residue adopted an unfavorable backbone conformation, with the energetic penalty being offset by a favorable aromatic interaction between proximal molecules in the crystal. NMR spectroscopy studies provided evidence for the maintenance of folded structures in solution in these αβ-hybrid sequences.

Combined Electron Transfer Dissociation–Collision-Induced Dissociation Fragmentation in the Mass Spectrometric Distinction of Leucine, Isoleucine, and Hydroxyproline Residues in Peptide Natural Products

Abstract: Distinctions between isobaric residues have been a major challenge in mass spectrometric peptide sequencing. Here, we propose a methodology for distinction among isobaric leucine, isoleucine, and hydroxyproline, a commonly found post-translationally modified amino acid with a nominal mass of 113 Da, through a combined electron transfer dissociation-collision-induced dissociation approach. While the absence of c and z(center dot) ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Delta m = 43 Da) or ethyl radicals (Delta m = 29 Da), through collisional activation of z(center dot) radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific e ions, corresponding to the Yyy-Leu or -Ile segments, are absent or of low intensity. This tandem mass spectrometric (MSn) method has been successfully implemented in a liquid chromatography MSn platform to determine the identity of 23 different isobaric residues from a mixture of five different peptides. The approach is convenient for distinction of isobaric residues from any crude peptide mixture, typically encountered in natural peptide libraries or proteomic analysis.

NMR analysis of cross strand aromatic interactions in an 8 residue hairpin and a 14 residue three stranded β-sheet peptide.

Abstract: Cross strand aromatic interactions between a facing pair of phenylalanine residues in antiparallel β-sheet structures have been probed using two structurally defined model peptides. The octapeptide Boc-LFVDPLPLFV-OMe (peptide 1) favors the β-hairpin conformation nucleated by the type II′ β-turn formed by the DPro-LPro segment, placing Phe2 and Phe7 side chains in proximity. Two centrally positioned DPro-LPro segments facilitate the three stranded β-sheet formation in the 14 residue peptide Boc-LFVDPLPLFVADPLPLFV-OMe (peptide 2) in which the Phe2/Phe7 orientations are similar to that in the octapeptide. The anticipated folded conformations of peptides 1 and 2 are established by the delineation of intramolecularly hydrogen bonded NH groups and by the observation of specific cross strand NOEs. The observation of ring current shifted aromatic protons is a diagnostic of close approach of the Phe2 and Phe7 side chains. Specific assignment of aromatic proton resonances using HSQC and HSQC-TOCSY methods allow an ...

The Structural Characterization of Folded Peptides Containing the Conformationally Constrained beta-Amino Acid Residue beta(2,2)Ac(6)c

Abstract: Backbone alkylation has been shown to result in a dramatic reduction in the conformational space that is sterically accessible to a-amino acid residues in peptides. By extension, the presence of geminal dialkyl substituents at backbone atoms also restricts available conformational space for beta and ? residues. Five peptides containing the achiral beta 2,2-disubstituted beta-amino acid residue, 1-(aminomethyl)cyclohexanecarboxylic acid (beta 2,2Ac6c), have been structurally characterized in crystals by X-ray diffraction. The tripeptide Boc-Aib-beta 2,2Ac6c-Aib-OMe (1) adopts a novel fold stabilized by two intramolecular H-bonds (C11 and C9) of opposite directionality. The tetrapeptide Boc-Aib-beta 2,2Ac6c]2-OMe (2) and pentapeptide Boc-Aib-beta 2,2Ac6c]2-Aib-OMe (3) form short stretches of a hybrid a beta C11 helix stabilized by two and three intramolecular H-bonds, respectively. The structure of the dipeptide Boc-Aib-beta 2,2Ac6c-OMe (5) does not reveal any intramolecular H-bond. The aggregation pattern in the crystal provides an example of an extended conformation of the beta 2,2Ac6c residue, forming a polar sheet like H-bond. The protected derivative Ac-beta 2,2Ac6c-NHMe (4) adopts a locally folded gauche conformation about the C beta?Ca bonds (?=-55.7 degrees). Of the seven examples of beta 2,2Ac6c residues reported here, six adopt gauche conformations, a feature which promotes local folding when incorporated into peptides. A comparison between the conformational properties of beta 2,2Ac6c and beta 3,3Ac6c residues, in peptides, is presented. Backbone torsional parameters of H-bonded a beta/beta a turns are derived from the structures presented in this study and earlier reports.

Characterization of bent helical conformations in polymorphic forms of a designed 18-residue peptide containing a central Gly-Pro segment.

Abstract: An 18-residue sequence Boc-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Gly-Pro-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe (UK18) was designed to examine the effect of introducing a Gly-Pro segment into the middle of a potentially helical peptide. The crystal structures of two polymorphic forms yielded a view of the conformation of three independent molecules. Form 1 (space group P212121, a = 14.620A; b = 26.506A, c = 28.858A, Z = 4) has one molecule in the asymmetric unit, with one cocrystallized water molecule. Form 2 (space group P212121, a = 9.696A; b = 19.641A, c = 114.31A, Z = 8) has two molecules in the asymmetric unit with four cocrystallized water molecules. In Form 1, residues 1 to 18 adopt ϕ,ψ values that lie in the right-handed helical (αR) region of the Ramachandran map. Two residues, Leu (8) (ϕ = −92.0°, ψ = −7.5°) and Leu (17) (ϕ = −94.7°, ψ = −1.7°) adopt conformations that deviate significantly from helical values. In Form 2, molecule A, residues 2 to 16 lie in the αR region of ϕ,ψ space, with Leu (8) (ϕ = −94.9°, ψ = −2.9°) deviating significantly from helical values. Aib (1) and Aib (18) adopt left-handed (αL) helical conformation. Significant distortion is observed at Leu (17) (ϕ = −121.3°, ψ = −31.3°). Molecule B, Form 2, adopts a right-handed helix over residues 1 to 17. In all three molecules, a distinct bend in the helix is observed, with the bend angle values varying from 40.8° to 58.9°. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 76–86, 2012.

Helix and hairpin nucleation in short peptides using centrally positioned conformationally constrained dipeptide segments

Abstract: The effect of incorporation of a centrally positioned Ac(6)c-Xxx segment where Xxx = (L)Val/(D)Val into a host oligopeptide composed of l-amino acid residues has been investigated. Studies of four designed octapeptides Boc-Leu-Phe-Val-Ac(6)c-Xxx-Leu-Phe-Val-OMe (Xxx = (D)Val 1, (L)Val 2) Boc-Leu-Val-Val-Ac(6)c-Xxx-Leu-Val-Val-OMe (Xxx = (D)Val 3, (L)Val 4) are reported. Diagnostic nuclear Overhouse effects characteristic of hairpin conformations are observed for Xxx = (D)Val peptides (1 and 3) while continuous helical conformation characterized by sequential N(i)H ↔ N(i+1)H NOEs are favored for Xxx = (L)Val peptides (2 and 4) in methanol solutions. Temperature co-efficient of NH chemical shifts are in agreement with distinctly different conformational preferences upon changing the configuration of the residue at position 5. Crystal structures of peptides 2 and 4 (Xxx = (L)Val) establish helical conformations in the solid state, in agreement with the structures deduced from NMR data. The results support the design principle that centrally positioned type I β-turns may be used to nucleate helices in short peptides, while type I'β-turns can facilitate folding into β-hairpins.

beta-Turn Analogues in Model a ss-Hybrid Peptides: Structural Characterization of Peptides Containing ss 2,2Ac6c and ss 3,3Ac6c Residues

Abstract: The effect of gem-dialkyl substituents on the backbone conformations of beta-amino acid residues in peptides has been investigated by using four model peptides: Boc-Xxx-beta 2,2Ac6c(1-aminomethylcyclohexanecarboxylic acid)-NHMe (Xxx=Leu (1), Phe (2); Boc=tert-butyloxycarbonyl) and Boc-Xxx-beta 3,3Ac6c(1-aminocyclohexaneacetic acid)-NHMe (Xxx=Leu (3), Phe (4)). Tetrasubstituted carbon atoms restrict the ranges of stereochemically allowed conformations about flanking single bonds. The crystal structure of Boc-Leu-beta 2,2Ac6c-NHMe (1) established a C11 hydrogen-bonded turn in the a beta-hybrid sequence. The observed torsion angles (a(similar to-60 degrees, similar to-30 degrees), beta(similar to-90 degrees, similar to 60 degrees, similar to-90 degrees)) corresponded to a C11 helical turn, which was a backbone-expanded analogue of the type III beta turn in aa sequences. The crystal structure of the peptide Boc-Phe-beta 3,3Ac6c-NHMe (4) established a C11 hydrogen-bonded turn with distinctly different backbone torsion angles (a(similar to-60 degrees, similar to 120 degrees), beta(similar to 60 degrees, ?60 degrees, similar to-60 degrees)), which corresponded to a backbone-expanded analogue of the type II beta turn observed in aa sequences. In peptide 4, the two molecules in the asymmetric unit adopted backbone torsion angles of opposite signs. In one of the molecules, the Phe residue adopted an unfavorable backbone conformation, with the energetic penalty being offset by a favorable aromatic interaction between proximal molecules in the crystal. NMR spectroscopy studies provided evidence for the maintenance of folded structures in solution in these a beta-hybrid sequences.