C₁₁/C₉ Helices in Crystals of αβ Hybrid Peptides and Switching Structures between Helix Types by Variation in the α‑Residue

Close-packed helices with mixed hydrogen bond directionality are unprecedented in the structural chemistry of α-polypeptides. While NMR studies in solution state provide strong evidence for the occurrence of mixed helices in (ββ)_<i>n</i> and (αβ)_<i>n</i> sequences, limited information is currently available in crystals. The peptide structures presented show the occurrence of C₁₁/C₉ helices in (αβ)_<i>n</i> peptides. Transitions between C₁₁ and C₁₁/C₉ helices are observed upon varying the α-amino acid residue

Cone Snail Glutaminyl Cyclase Sequences from Transcriptomic Analysis and Mass Spectrometric Characterization of Two Pyroglutamyl Conotoxins

The post-translational modification of N-terminal glutamine (Q) to a pyroglutamyl (Z) residue is observed in the conotoxins produced by marine cone snails. This conversion requires the action of the enzyme glutaminyl cyclase (QC). Four complete QC sequences from the species <i>C. araneosus</i>, <i>C. frigidus, C. litteratus</i>, and <i>C. monile</i> and two partial sequences from <i>C. amadis</i> and <i>C. miles</i> have been obtained by analysis of transcriptomic data. Comparisons with mammalian enzyme sequences establish a high level of identity and complete conservation of functional active site residues, including a cluster of hydrogen-bonded acidic side chains. Mass spectrometric analysis of crude venom samples coupled to conotoxin precursor protein sequences obtained from transcriptomic data establishes the presence of pyroglutamyl conotoxins in the venom of <i>C. frigidus</i> and <i>C. amadis</i>. The <i>C. frigidus</i> peptide belongs to the M superfamily, with cysteine framework III, whereas the <i>C. amadis</i> peptide belongs to the divergent superfamily with cysteine framework VI/VII. Additionally, gamma carboxylation of glutamic acid and hydroxylation of proline are observed in the <i>C. frigidus</i> peptide. Mass spectral data are available via ProteomeXchange with identifier PXD009006

NMR Analysis of Cross Strand Aromatic Interactions in an 8 Residue Hairpin and a 14 Residue Three Stranded β‑Sheet Peptide

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-LFV^DP^LPLFV-OMe (peptide <b>1</b>) 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-LFV^DP^LPLFVA^DP^LPLFV-OMe (peptide <b>2</b>) in which the Phe2/Phe7 orientations are similar to that in the octapeptide. The anticipated folded conformations of peptides <b>1</b> and <b>2</b> 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 analysis of interproton NOEs between the spatially proximate aromatic rings. This approach facilitates specific assignments in systems containing multiple aromatic rings in spectra at natural abundance. Evidence is presented for a dynamic process which invokes a correlated conformational change about the C^α-C^β(χ^<b>1</b>) bond for the pair of interacting Phe residues. NMR results suggest that aromatic ring orientations observed in crystals are maintained in solution. Anomalous temperature dependence of ring current induced proton chemical shifts suggests that solvophobic effects may facilitate aromatic ring clustering in apolar solvents

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

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 <i>c</i> and <i>z</i>^<i>•</i> ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Δ<i>m</i> = 43 Da) or ethyl radicals (Δ<i>m</i> = 29 Da), through collisional activation of <i>z</i> radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific <i>z</i>^<i>•</i> ions, corresponding to the Yyy–Leu or −Ile segments, are absent or of low intensity. This tandem mass spectrometric (MS^<i>n</i>) method has been successfully implemented in a liquid chromatography–MS^<i>n</i> 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

Temperature-Induced Reversible First-Order Single Crystal to Single Crystal Phase Transition in Boc‑γ⁴(<i>R</i>)Val-Val-OH: Interplay of Enthalpy and Entropy

Crystals of Boc-γ⁴(<i>R</i>)­Val-Val-OH undergo a reversible first-order single crystal to single crystal phase transition at <i>T</i>_c ≈ 205 K from the orthorhombic space group <i>P</i>22₁2₁ (<i>Z</i>′ = 1) to the monoclinic space group <i>P</i>2₁ (<i>Z</i>′ = 2) with a hysteresis of ∼2.1 K. The low-temperature monoclinic form is best described as a nonmerohedral twin with ∼50% contributions from its two components. The thermal behavior of the dipeptide crystals was characterized by differential scanning calorimetry experiments. Visual changes in birefringence of the sample during heating and cooling cycles on a hot-stage microscope with polarized light supported the phase transition. Variable-temperature unit cell check measurements from 300 to 100 K showed discontinuity in the volume and cell parameters near the transition temperature, supporting the first-order behavior. A detailed comparison of the room-temperature orthorhombic form with the low-temperature (100 K) monoclinic form revealed that the strong hydrogen-bonding motif is retained in both crystal systems, whereas the non-covalent interactions involving side chains of the dipeptide differ significantly, leading to a small change in molecular conformation in the monoclinic form as well as a small reorientation of the molecules along the <i>ac</i> plane. A rigid-body thermal motion analysis (translation, libration, screw; correlation of translation and libration) was performed to study the crystal entropy. The reversible nature of the phase transition is probably the result of an interplay between enthalpy and entropy: the low-temperature monoclinic form is enthalpically favored, whereas the room-temperature orthorhombic form is entropically favored

C₁₂ Helices in Long Hybrid (αγ)_<i>n</i> Peptides Composed Entirely of Unconstrained Residues with Proteinogenic Side Chains

Unconstrained γ⁴ amino acid residues derived by homologation of proteinogenic amino acids facilitate helical folding in hybrid (αγ)_<i>n</i> sequences. The C₁₂ helical conformation for the decapeptide, Boc-[Leu-γ⁴(<i>R</i>)­Val]₅-OMe, is established in crystals by X-ray diffraction. A regular C₁₂ helix is demonstrated by NMR studies of the 18 residue peptide, Boc-[Leu-γ⁴(<i>R</i>)­Val]₉-OMe, and a designed 16 residue (αγ)_<i>n</i> peptide, incorporating variable side chains. Unconstrained (αγ)_<i>n</i> peptides show an unexpectedly high propensity for helical folding in long polypeptide sequences

Revisiting the Burden Borne by Fumarase: Enzymatic Hydration of an Olefin

Fumarate hydratase (FH) is a remarkable catalyst that decreases the free energy of the catalyzed reaction by 30 kcal mol–1, much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, which have different folds, yet catalyze the same reversible hydration/dehydration reaction of the dicarboxylic acids fumarate/malate, with equal efficiencies. Using class-I FH from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj) as a model along with comparative analysis with the only other available class-I FH structure from Leishmania major (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes. The structure of MjFH apo-protein has been determined, revealing that large intersubunit rearrangements occur across apo- and holo-protein forms, with a largely preorganized active site for substrate binding. Site-directed mutagenesis of active site residues, kinetic analysis, and computational studies, including density functional theory (DFT) and natural population analysis, together show that residues interacting with the carboxylate group of the substrate play a pivotal role in catalysis. Our study establishes that an electrostatic network at the active site of class-I FH polarizes the substrate fumarate through interactions with its carboxylate groups, thereby permitting an easier addition of a water molecule across the olefinic bond. We propose a mechanism of catalysis in FH that occurs through transition-state stabilization involving the distortion of the electronic structure of the substrate olefinic bond mediated by the charge polarization of the bound substrate at the enzyme active site

Temperature-Induced Reversible First-Order Single Crystal to Single Crystal Phase Transition in Boc‑γ⁴(<i>R</i>)Val-Val-OH: Interplay of Enthalpy and Entropy

Crystals of Boc-γ⁴(<i>R</i>)­Val-Val-OH undergo a reversible first-order single crystal to single crystal phase transition at <i>T</i>_c ≈ 205 K from the orthorhombic space group <i>P</i>22₁2₁ (<i>Z</i>′ = 1) to the monoclinic space group <i>P</i>2₁ (<i>Z</i>′ = 2) with a hysteresis of ∼2.1 K. The low-temperature monoclinic form is best described as a nonmerohedral twin with ∼50% contributions from its two components. The thermal behavior of the dipeptide crystals was characterized by differential scanning calorimetry experiments. Visual changes in birefringence of the sample during heating and cooling cycles on a hot-stage microscope with polarized light supported the phase transition. Variable-temperature unit cell check measurements from 300 to 100 K showed discontinuity in the volume and cell parameters near the transition temperature, supporting the first-order behavior. A detailed comparison of the room-temperature orthorhombic form with the low-temperature (100 K) monoclinic form revealed that the strong hydrogen-bonding motif is retained in both crystal systems, whereas the non-covalent interactions involving side chains of the dipeptide differ significantly, leading to a small change in molecular conformation in the monoclinic form as well as a small reorientation of the molecules along the <i>ac</i> plane. A rigid-body thermal motion analysis (translation, libration, screw; correlation of translation and libration) was performed to study the crystal entropy. The reversible nature of the phase transition is probably the result of an interplay between enthalpy and entropy: the low-temperature monoclinic form is enthalpically favored, whereas the room-temperature orthorhombic form is entropically favored

Unconstrained Homooligomeric γ‑Peptides Show High Propensity for C₁₄ Helix Formation

Monosubstituted γ⁴-residues (γ⁴Leu, γ⁴Ile, and γ⁴Val) form helices even in short homooligomeric sequences. C₁₄ helix formation is established by X-ray diffraction in homooligomeric (γ)_<i>n</i> tetra-, hexa- and decapeptide sequences demonstrating the high propensity of γ residues, with proteinogenic side chains, to adopt locally folded conformations

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

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 μ-conotoxin KIIIA, which was predicted originally to have a [C1–C9,C2–C15,C4–C16] disulfide pattern based on homology with closely related μ-conotoxins. The two major isomers of synthetic μ-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_V1.2 (<i>K</i>_d values of 5 and 230 nM, respectively). The solution structure for μ-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 μ-KIIIA structure calculated with the incorrect [C1–C9,C2–C15,C4–C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as μ-KIIIA, and both blocked Na_V1.2 (<i>K</i>_d values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA and μ-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 μ-KIIIA in the venom of <i>Conus kinoshitai</i>; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail’s repertoire of active peptides