There is a dogma in molecular biology that biological functions of peptides are determined by their structure ("function" code), coded in their primary structure ("structure" code). This work describes a new approach that attempts to elucidate these relationships by peptide topology design based on intriguing conformational properties of pipecolic acid based amino acids--like 1,2,3,4 tetrahydroisoquinoline (Tic). Opioid peptides, owing to the heterogeneity of opioid receptors, display a wide variety of physiological actions. The mu opioid receptor selective octapeptide I (D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂) is a model compound for topographical modifications induced by sequential substitutions by Tic residue. Thus, the closely related peptides I and II (Gly-D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂, obtained by coupling Gly residue to I) have contrasting affinities for the mu opioid receptor (IC₅₀ = 1.2 and 278 nM, respectively). Conformational analysis of I and II by means of 1D and 2D ¹H NMR spectroscopy allowed to determine dramatic differences in the side chain orientation of D-Tic in both peptides and to propose features of the bioactive conformation. The extended conformation of I (due to g(-) side chain conformation of D-Tic) is well recognized by the mu receptor in contrast to the folded conformation of II (due to a g(+) side chain conformation of D-Tic¹, that places the aromatic ring on the opposite side of the molecule), which is not. Peptide III (D-Phe-Cys-Tic-D-Trp-Orn-Thr-Pen-Thr-NH₂), featuring replacement of Tyr³ by Tic³, binds very weakly to the mu opioid receptor, due to rotation of the Tic aromatic side chain to the opposite side of the molecule (Tic side chain is in a g(+) conformation again). As these substitutions conserve the conformation of the backbone, constrained cyclic amino acids (picolic acid derivatives) can modify the topography of the peptide in a predictable manner, and (in conjunction with biological data) disclose structural elements of bioactive conformations. The mechanisms of pipecolic acid side chain rotamer selection, will be discussed in the context of design principles.

Cyclic peptides possess desirable characteristics as potential pharmaceutical scaffolds. The cyanobactin family of cyclic peptide natural products boast diverse structures and bioactivity. Exemplars are the patellamides, which have attracted attention due to their ability to reverse the effects of multi-drug resistance in human leukemia cells. In addition to their macrocyclic architecture patellamides contain azol(in)e heterocycles and d-amino acids. This structural complexity makes them challenging targets for chemical synthesis. Understanding their biosynthesis will enable the development of a biotechnological ‘toolkit' for the synthesis of new pharmaceutical compounds. Patellamides are ribosomally-synthesised and post-translationally modified peptides (RiPPs) and much of their biosynthesis has been elucidated, however there are still elements of their biosynthesis that are not yet fully understood. PatA and PatG contain C-terminal domains of unknown function (DUFs). The crystal structure of PatG-DUF has been solved and subsequent to biochemical and biophysical investigation PatG-DUF was found not to constitute an essential part of the biotechnological ‘toolkit' and can be excluded from in vitro enzyme-based synthesis of cyanobactin-like cyclic peptides. The cyanobactin heterocyclases are able to introduce heterocycles into a peptide backbone, seemingly irrespective of the neighbouring residues; however a molecular rational governing substrate recognition is unknown. Additionally the mechanism of heterocyclisaton is disputed. Analysis of crystal structures of LynD in complex with cofactor and substrate (solved by Dr Jesko Koehnke) enabled the active site and substrate recognition site to be located. A new mechanism for heterocyclisation has been proposed. Guided by the substrate recognition observed in complex structures a constituently active heterocyclase (AcLynD) has been engineered, which is able to process short, leaderless peptide substrates. Epimerisation in cyanobactin biosynthesis is believed to be spontaneous, but its precise timing is uncertain. NMR analysis of selectively labelled peptide substrates processed by the modifying enzymes, identified epimerisation to be spontaneous on the macrocycle, regardless of whether the neighbouring heterocycles have been oxidised. A one-pot in vitro synthesis of cyanobactins has been developed, and employed to create a number of patellamide D analogues to ascertain structural-activity relationships.

Cyclic peptides have potential as scaffolds for novel pharmaceuticals, however their chemical synthesis can be challenging and as such natural sources are often explored. Several species of cyanobacteria produce a family of cyclic peptides, the cyanobactins, through the ribosomal synthesis of precursor peptides and post-translational tailoring. The patellamides, a member of the cyanobactin family, are cyclic octapeptides containing D-stereo centres and heterocyclised amino acids. A single gene cluster, patA - patG, contains the genes for the expression of the precursor peptide and the enzymes responsible for post-translational modifications including a heterocyclase, protease, macrocyclase and oxidase. Biochemical and structural analysis on the patellamide and related cyanobactin pathways has been carried out. The crystal structure of PatF, a proposed prenyl transferase, has been determined, highlighting that it is likely evolutionary inactive due to changes to key residues when compared to active homologues. This is in agreement with the knowledge that no naturally prenylated patellamides have been discovered to date. The crystal structure of the macrocyclase domain of PatG has been determined in complex with a substrate analogue peptide. The structure, together with biochemical analysis has allowed a mechanism of macrocyclisation to be proposed, confirming the requirement of a specific substrate conformation to enable macrocyclisation. Using isolated enzymes from the patellamide and related pathways, a small scale library of macrocycles made up of diverse sequences has been created in vitro and characterised by mass spectrometry and in certain cases NMR. In order to further enhance diversity, macrocycles containing unnatural amino acids have been created using three approaches; SeCys derived precursor peptides, intein-mediated peptide ligation and pEVOL amber codon technology. Finally, two oxidase enzymes from cyanobactin pathways have been purified, characterised and confirmed active for thiazoline oxidation. Native X-ray datasets on crystals of the oxidase CyaGox have been collected and phasing trials are on-going.

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Indiana University-Purdue University Indianapolis (IUPUI) / Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4µg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4µg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability.