Mark Miskolzie

Accessing zinc monohydride cations through coordinative interactions

We present isolable examples of formal zinc hydride cations supported by N-heterocyclic carbene (NHC) donors, and investigate the dual electrophilic and nucleophilic (hydridic) character of the encapsulated [ZnH](+) units by computational methods and preliminary hydrosilylation catalysis.

Experimental investigations of a partial Ru-O bond during the metal-ligand bifunctional addition in Noyori-type enantioselective ketone hydrogenation.

The transition state for the metal-ligand bifunctional addition step in Noyoris enantioselective ketone hydrogenation was investigated using intramolecular trapping experiments. The bifunctional addition between the Ru dihydride trans-[Ru((R)-BINAP)(H)(2)((R,R)-dpen)] and the hydroxy ketone 4-HOCH(2)C(6)H(4)(CO)CH(3) at -80 °C exclusively formed the corresponding secondary ruthenium alkoxide trans-[Ru((R)-BINAP)(H)(4-HOCH(2)C(6)H(4)CH(CH(3))O)((R,R)-dpen)]. Combined with the results of control experiments, this observation provides strong evidence for the formation of a partial Ru-O bond in the transition state.

Solution structures of the linear leaderless bacteriocins enterocin 7A and 7B resemble carnocyclin A, a circular antimicrobial peptide.

Leaderless bacteriocins are a class of ribosomally synthesized antimicrobial peptides that are produced by certain Gram-positive bacteria without an N-terminal leader section. These bacteriocins are of great interest due to their potent inhibition of many Gram-positive organisms, including food-borne pathogens such as Listeria and Clostridium spp. We now report the NMR solution structures of enterocins 7A and 7B, leaderless bacteriocins recently isolated from Enterococcus faecalis 710C. These are the first three-dimensional structures to be reported for bacteriocins of this class. Unlike most other linear Gram-positive bacteriocins, enterocins 7A and 7B are highly structured in aqueous conditions. Both peptides are primarily α-helical, adopting a similar overall fold. The structures can be divided into three separate α-helical regions: the N- and C-termini are both α-helical, separated by a central kinked α-helix. The overall structures bear an unexpected resemblance to carnocyclin A, a 60-residue peptide that is cyclized via an amide bond between the C- and N-termini and has a saposin fold. Because of synergism observed for other two-peptide leaderless bacteriocins, it was of interest to probe possible binding interactions between enterocins 7A and 7B. However, despite synergistic activity observed between these peptides, no significant binding interaction was observed based on NMR and isothermal calorimetry.

Solution Structure of Acidocin B, a Circular Bacteriocin Produced by Lactobacillus acidophilus M46.

ABSTRACT Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four α-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ∼7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four α-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.

Substitution of a Conserved Disulfide in the Type IIa Bacteriocin, Leucocin A, with L-Leucine and L-Serine Residues: Effects on Activity and Three-Dimensional Structure

Many lactic acid bacteria (LAB) are known to produce bacteriocins, a class of ribosomally synthesized antimicrobial peptides. Several of these bacteriocins, such as nisin A and pediocin PA1, exhibit activity against food spoilage organisms at nanomolar concentrations. Thus, these peptides are commonly used as food preservatives in dairy products and processed meat. Leucocin A is a bacteriocin produced by Leuconostoc gelidum UAL 187, an LAB strain that was isolated from vacuum-packaged meat. This peptide was the first type IIa bacteriocin whose sequence was reported. It can inhibit Listeria monocytogenes, Enterococcus faecalis, and a variety of LAB. Leucocin A is 37 residues long, and features a disulfide bond between cysteine 9 and cysteine 14 (Scheme 1 A). It shares this highly con-

Nuclear Magnetic Resonance Solution Structures of Lacticin Q and Aureocin A53 Reveal a Structural Motif Conserved among Leaderless Bacteriocins with Broad-Spectrum Activity

Lacticin Q (LnqQ) and aureocin A53 (AucA) are leaderless bacteriocins from Lactococcus lactis QU5 and Staphylococcus aureus A53, respectively. These bacteriocins are characterized by the absence of an N-terminal leader sequence and are active against a broad range of Gram-positive bacteria. LnqQ and AucA consist of 53 and 51 amino acids, respectively, and have 47% identical sequences. In this study, their three-dimensional structures were elucidated using solution nuclear magnetic resonance and were shown to consist of four α-helices that assume a very similar compact, globular overall fold (root-mean-square deviation of 1.7 Å) with a highly cationic surface and a hydrophobic core. The structures of LnqQ and AucA resemble the shorter two-component leaderless bacteriocins, enterocins 7A and 7B, despite having low levels of sequence identity. Homology modeling revealed that the observed structural motif may be shared among leaderless bacteriocins with broad-spectrum activity against Gram-positive organisms. The elucidated structures of LnqQ and AucA also exhibit some resemblance to circular bacteriocins. Despite their similar overall fold, inhibition studies showed that LnqQ and AucA have different antimicrobial potency against the Gram-positive strains tested, suggesting that sequence disparities play a crucial role in their mechanisms of action.

In Vivo Screening Identifies a Highly Folded β-Hairpin Peptide with a Structured Extension

The conventional approach for developing folded peptides involves chemical synthesis of systematically modified peptide variants and individual characterization by circular dichroism and NMR spectroscopy. The use of synthetic peptides and reliance on low-throughput characterization techniques necessarily restricts the sequence diversity that can be explored, though efforts have been made to overcome this limitation. We have recently described an approach that enables us to asses the ability of peptides to fold into b-hairpins in the cytoplasm of live cells. Our strategy entails the recombinant expression of a peptide gene fused in frame between flanking genes encoding a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). If a particular peptide sequence adopts a folded structure, CFP and YFP are brought into closer proximity and exhibit a higher efficiency of fluorescence resonance energy transfer (FRET). Higher FRET efficiency enhances the YFP (acceptor) fluorescence at the expense of the CFP (donor) fluorescence. Imaging of plates harboring colonies of transformed bacteria provides the YFP and CFP fluorescence emission intensities for each colony, and thus peptides that are highly folded in vivo can be distinguished from those that are not. As will be described in this manuscript, this FRET-based approach also provides a versatile method for screening large libraries of peptide sequences for highly structured variants. We have applied this strategy to the development of a version of a “tryptophan zipper” (trpzip)-type b-hairpin with a structured extension. Trpzips are a class of highly folded b-hairpins that are defined by the presence of cross-strand diagonally oriented Trp/Trp pairs on one face of the hairpin. The minimal and highly stable trpzip structure has emerged as a preferred model system for computational and experimental studies of protein folding. We have proposed that trpzip-type peptides (or tandem fusions of such peptides) could serve as a minimal protein scaffold for molecular recognition in the cytoplasm of live cells. With this goal in mind, we sought to employ our screening strategy to identify a candidate trpziptype peptide with high fold stability in vivo. A similar approach has previously been used to increase the thermal stability of engineered immunoglobulin VL domains. [15] The template for our initial “extended trpzip” library was a 20-mer version of the highly folded 16-mer trpzip HP5W4. The 20-mer contained two additional pairs of residues, one random and one threonine, genetically inserted after what would otherwise have been the second and 14th residues of HP5W4. The sequence of the resulting “peptide” portion of the 400-member protein library was K KXTWTWNP ATG KWTWTXQE ; here X represents all 20 amino acids, and the residues inserted relative to HP5W4 are underlined. Assuming b-strand conformation, the randomized positions would be directed towards the face of HP5W4 that harbors the interdigitated Trp side chains. Escherichia coli was transformed with the gene library, and ~6F10 colonies on 10 Petri dishes were subject to fluorescence imaging in order to identify those exhibiting the highest ratio of YFP-to-CFP fluorescence emission. We have previously demonstrated the ability of this imaging system to reliably distinguish colonies that express FRET constructs of various FRET efficiencies. The fluorescent brightness of each colony, considered commensurate with peptide solubility, was determined by direct excitation and imaging of YFP. Five individual colonies that exhibited both a high ratio of YFP-to-CFP fluorescence emission and high brightness were cultured overnight, and the plasmid DNA was purified. DNA sequencing revealed a striking consensus at the randomized positions: in all five sequences, position 3 was Trp and position 18 was either Lys or Arg (Table 1). This consensus sequence was used as the new

NMR Conformational Studies of Micelle-Bound Orexin-B: A Neuropeptide Involved in the Sleep/Awake Cycle and Feeding Regulation

Abstract The preferred conformation of orexin-B, an orphan G-protein coupled receptor agonist (the human sequence is RSGPPGLQGRLQRLLQASGNHAAGILTM-NH2) has been determined by 1H and 13C 2D NMR spectroscopy and molecular modeling. Orexin-B has been implicated in sleep-wakefulness and feeding regulation. The membrane mimetic, sodium dodecylsulphate-d25 (SDS), was used to mimic a physiological environment for the peptide. The secondary structure of orexin-B in SDS consists of two helical sections; helix I spans Leu7 to Ser18 and helix II spans Ala22 to Leu26. Helices I and II are believed to be involved in membrane binding, as is supported by the results of the spin label studies with 5-doxylstearic acid. Lee et al. (Eur. J. Biochem. 266, 831–839 (1999)) determined the [Phe1]-orex- in-B conformation in water solution by NMR and showed that helix II extends from Ala23 to Met28. The C-terminal dipeptide, Thr27-Met28, is unstructured is SDS, whereas in water it forms the end of helix II. The lack of apparent structure for Thr27-Met28 in SDS allows the dipeptide to have conformational freedom to interact with the receptor. The conformation of orexin-B can now be used to explain the Ala substitution mutagenesis experiments and the D-amino acid substitution experiments (S. Asahi et al, Bioorg. Med. Chem. Lett. 13, 111–113, 2003). Asahi et al. have shown that Ala substitution from Gly24 to Met28 or D- amino acid substitution from Ala23 to Met28 causes a significant reduction in the potency of orexin-B for both OX1R and OX2R receptors. We postulate that helix II is involved in membrane recognition, and its binding to the membrane is essential for Thr27-Met28 to adopt the correct receptor-binding conformation.

The NMR-Derived Conformation of Orexin-A: An Orphan G-Protein Coupled Receptor Agonist Involved in Appetite Regulation and Sleep

Abstract The conformation of orexin-A, an orphan G-protein coupled receptor agonist (the human sequence is: has been determined when bound to sodium dodecylsulphate-d25 (SDS) micelles by 1H and 13C NMR and molecular modeling. Orexin-A has been implicated in sleep-wakefulness and feeding regulation. The conformational preference of orexin-A consists of a short helical section, involving Asp5 to Gln9 that makes up helix I, followed by a bend from Lys10 to Ser13. Residues Leu16 to Gly22 make up helix II. The conformation of orexin-A can now be used to explain the results of earlier Ala substitution mutagenesis experiments (J. G. Darker, et al. Bioorg. Med. Chem. Lett. 11, 737–740 (2001); S. Ammoun, et al. J. Pharmacol. Expt. Ther. 305, 507–514 (2003)). Darker et al., working with orexin-A (15–33) amide, observed a significant drop in functional potency at the OX1R receptor when Leu16, Leu19, Leu20, His26, Gly29, Ile30, Leu31, Thr32, and Leu33 were replaced by Ala. Ammoun et al. identified three areas of interest, which were the same for OX1R and OX2 R receptors, as amino acids 15–17, 20 and 25–26 with the most marked reduction in activity being produced by the replacement of Leu20 by Ala. We suggest that Leu16, Leu19, and Leu20, which are in helix II, are likely responsible for binding orexin-A to the surface of the micelle.

NMR Conformational Analyses on (des-bromo) Neuropeptide B [1–23] and Neuropeptide W [1–23]: The Importance of α-helices, a Cation-π Interaction and a β-Turn

Abstract The preferred conformations of the orphan G-protein coupled receptor agonists (des-bromo) neuropeptide B [1–23] and neuropeptide W [1–23], referred to as NPB and NPW, have been determined by 1H NMR, CD, and molecular modeling. The sequences of NPB and NPW are WYKPAAGHSSYSVGRAAGLLSGL and WYKHVASPRYHTVGRAAGLLMGL, respectively. These are hypothalamic peptides that exert their biological actions on GPR7 and GPR8 receptors. Micellar solutions using the membrane mimetic, sodium dodecylsulphate-d25 (SDS), were used to mimic a physiological environment for the peptides. The secondary structure of NPB consists of a type II β-turn involving residues Lys3 to Ala6. The C-teminal region of NPB exists in a conformational equilibrium between different secondary structures, including an a-helix from residues Arg15 to Ser21, and a 310-helix from residues Ser12 to Ser21. The N-teminus of NPW exhibits a cation-π interaction between the Lys3 side chain and the quadrupole moment of the Trp1 indole group. At the C-temiinus of NPW, a well-defined α-helical conformation exists from Arg15 to Met21. As NPB and NPW have 91% sequence homology from residues Val13 to Leu23, with only residue 21 differing between the two peptides, the similar C-terminal secondary structures of these two peptides are consistent with the sequences. This is supported by the similar CD spectra. The different secondary structures at the N-termini for NPB and NPW point to the importance of the N-terminus in receptor binding. This is consistent with the work of Fujii et al. [J. Biol. Chem. 277, 34010–34016 (2002)] who observed that iodination of the NPB Tyr2 resulted in decreased agonistic activity at GPR7. In addition, Tanaka et al. [Proc. Natl. Acad. Sci. USA 100, 6251–6256 (2003)] showed that deletion of Tip1 from NPB or NPW drastically decreased activity at GPR7 for NPB and GPR7 and GPR8 for NPW. Therefore, we postulate that the N-terminus is involved in membrane recognition and receptor binding.