E. Neil G. Marsh

Role of zinc in human islet amyloid polypeptide aggregation

Human Islet Amyloid Polypeptide (hIAPP) is a highly amyloidogenic protein found in islet cells of patients with type II diabetes. Because hIAPP is highly toxic to beta-cells under certain conditions, it has been proposed that hIAPP is linked to the loss of beta-cells and insulin secretion in type II diabetics. One of the interesting questions surrounding this peptide is how the toxic and aggregation prone hIAPP peptide can be maintained in a safe state at the high concentrations that are found in the secretory granule where it is stored. We show here zinc, which is found at millimolar concentrations in the secretory granule, significantly inhibits hIAPP amyloid fibrillogenesis at concentrations similar to those found in the extracellular environment. Zinc has a dual effect on hIAPP fibrillogenesis: it increases the lag-time for fiber formation and decreases the rate of addition of hIAPP to existing fibers at lower concentrations, while having the opposite effect at higher concentrations. Experiments at an acidic pH which partially neutralizes the change in charge upon zinc binding show inhibition is largely due to an electrostatic effect at His18. High-resolution structures of hIAPP determined from NMR experiments confirm zinc binding to His18 and indicate zinc induces localized disruption of the secondary structure of IAPP in the vicinity of His18 of a putative helical intermediate of IAPP. The inhibition of the formation of aggregated and toxic forms of hIAPP by zinc provides a possible mechanism between the recent discovery of linkage between deleterious mutations in the SLC30A8 zinc transporter, which transports zinc into the secretory granule, and type II diabetes.

Oxygen-independent decarbonylation of aldehydes by cyanobacterial aldehyde decarbonylase: a new reaction of diiron enzymes.

The search for new biofuels has generated increased interest in biochemical pathways that produce hydrocarbons.[1] Although hydrocarbons are simple molecules, the biosynthesis of molecules that lack any chemical functional groups is surprisingly challenging.[2] Biochemical reactions that remove functionality, such as decarboxylations, dehydrations and reduction of double bonds, invariably rely on the presence of adjacent functional groups to stabilize unfavorable transition states. Enzymes involved in hydrocarbon biosynthesis are therefore of interest both for applications in biofuels production and because of the unusual and chemically difficult reactions they catalyze.[3] One enzyme that has attracted particular interest, is aldehyde decarbonylase (AD), which catalyzes the decarbonylation of long-chain fatty aldehydes, to the corresponding alkanes.[4]

Alternative pathways of human islet amyloid polypeptide aggregation distinguished by 19F nuclear magnetic resonance-detected kinetics of monomer consumption

Amyloid formation, a complex process involving many intermediate states, is proposed to be the driving force for amyloid-related toxicity in common degenerative diseases. Unfortunately, the details of this process have been obscured by the limitations in the methods that can follow this reaction in real time. We show that alternative pathways of aggregation can be distinguished by using (19)F nuclear magnetic resonance (NMR) to monitor monomer consumption along with complementary measurements of fibrillogenesis. The utility of this technique is demonstrated by tracking amyloid formation in the diabetes-related islet amyloid polypeptide (IAPP). Using this technique, we show IAPP fibrillizes without an appreciable buildup of nonfibrillar intermediates, in contrast to the well-studied Aβ and α-synuclein proteins. To further develop the usage of (19)F NMR, we have tracked the influence of the polyphenolic amyloid inhibitor epigallocatechin gallate (EGCG) on the aggregation pathway. Polyphenols have been shown to strongly inhibit amyloid formation in many systems. However, spectroscopic measurements of amyloid inhibition by these compounds can be severely compromised by background signals and competitive binding with extrinsic probes. Using (19)F NMR, we show that thioflavin T strongly competes with EGCG for binding sites on IAPP fibers. By comparing the rates of monomer consumption and fiber formation, we are able to show that EGCG stabilizes nonfibrillar large aggregates during fibrillogenesis.

Adenosyl radical: reagent and catalyst in enzyme reactions.

Adenosine is undoubtedly an ancient biological molecule that is a component of many enzyme cofactors: ATP, FADH, NAD(P)H, and coenzyme A, to name but a few, and, of course, of RNA. Here we present an overview of the role of adenosine in its most reactive form: as an organic radical formed either by homolytic cleavage of adenosylcobalamin (coenzyme B12, AdoCbl) or by single‐electron reduction of S‐adenosylmethionine (AdoMet) complexed to an iron–sulfur cluster. Although many of the enzymes we discuss are newly discovered, adenosines role as a radical cofactor most likely arose very early in evolution, before the advent of photosynthesis and the production of molecular oxygen, which rapidly inactivates many radical enzymes. AdoCbl‐dependent enzymes appear to be confined to a rather narrow repertoire of rearrangement reactions involving 1,2‐hydrogen atom migrations; nevertheless, mechanistic insights gained from studying these enzymes have proved extremely valuable in understanding how enzymes generate and control highly reactive free radical intermediates. In contrast, there has been a recent explosion in the number of radical‐AdoMet enzymes discovered that catalyze a remarkably wide range of chemically challenging reactions; here there is much still to learn about their mechanisms. Although all the radical‐AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes might be active in aerobic organisms including humans.

Using fluorous amino acids to modulate the biological activity of an antimicrobial peptide.

The emergence of bacterial strains resistant to most of the clinically useful antibiotics has provided the impetus to develop new classes of antibiotics that might combat bacterial resistance more effectively. Antimicrobial peptides (AMPs) are small peptides (typically 15–30 residues) that show promise as therapeutic agents against bacteria, fungi, and viruses. [1–3] Widely distributed in plants and animals, they form part of the innate immune system’s defense against microbes. Although highly diverse in sequence and structure, almost all AMPs share the property of being highly amphiphathic: one face of the peptide is hydrophobic and the other face presents a cluster of positively charged residues. [4, 5] AMPs function by disrupting bacterial membranes, [4] which contain predominantly negatively charged phospholipids. Eukaryotic membranes, which contain predominantly neutral phospholipids, are not targeted. Although promising as broad-spectrum antibiotics, AMPs are susceptible to proteolysis in vivo by endogenous or bacterial proteases, which can considerably diminish their effectiveness. Attempts to overcome this problem by increasing the dose of AMP often leads to toxic side effects, most notably lysis of red blood cells, which has been attributed to nonspecific hydrophobic interactions between the peptide and the eukaryotic cell membrane. [6, 7] Here we describe a strategy to overcome

Using Fluorous Amino Acids To Probe the Effects of Changing Hydrophobicity on the Physical and Biological Properties of the β-Hairpin Antimicrobial Peptide Protegrin-1†

Protegrins are potent members of the beta-hairpin-forming class of antimicrobial peptides. Key to their antimicrobial activity is their assembly into oligomeric structures upon binding to the bacterial membrane. To examine the relationship between the physicochemical properties of the peptide and its biological activity, we have synthesized variants of protegrin-1 in which key residues in the hydrophobic core, valine-14 and -16, are changed to leucine and to the extensively fluorinated analogue hexafluoroleucine. These substitutions have the effect of making the peptide progressively more hydrophobic while minimally perturbing the secondary structure. The leucine-containing peptide was significantly more active than wild-type protegrin against several common pathogenic bacterial strains, whereas the hexafluoroleucine-substituted peptide, in contrast, showed significantly diminished activity against several bacterial strains. Isothermal titration calorimetry measurements revealed significant changes in the interaction of the peptides binding to small unilamelar vesicles that mimic the lipid composition of the bacterial membrane. The binding isotherms for wild-type and leucine-substituted protegrins indicate that electrostatic interactions dominate the membrane-peptide interaction, whereas the isotherm for the hexafluoroleucine-substituted protegrin suggests a diminished electrostatic component to binding. Notably both of these substitutions appear to alter the stoichiometry of the lipid-peptide interaction, suggesting that these substitutions may stabilize oligomerized forms of protegrin that are postulated to be intermediates in the assembly of the beta-barrel membrane pore structure.

Adenosylcobalamin-dependent isomerases: New insights into structure and mechanism

Adenosylcobalamin-dependent isomerases catalyze a variety of chemically difficult 1,2-rearrangements that proceed through a mechanism involving free radical intermediates. These radicals are initially generated by homolysis of the cobalt-carbon bond of the coenzyme. Recently, the crystal structures of several of these enzymes have been solved, revealing two modes of coenzyme binding and highlighting the role of the protein in controlling the rearrangement of reactive substrate radical intermediates. Complementary data from kinetic, spectroscopic and theoretical studies have produced insights into the mechanism by which substrate radicals are generated at the active site, and the pathways by which they rearrange.

Using 19F NMR to Probe Biological Interactions of Proteins and Peptides

Fluorine is a valuable probe for investigating the interactions of biological molecules because of its favorable NMR characteristics, its small size, and its near total absence from biology. Advances in biosynthetic methods allow fluorine to be introduced into peptides and proteins with high precision, and the increasing sensitivity of NMR spectrometers has facilitated the use of (19)F NMR to obtain molecular-level insights into a wide range of often-complex biological interactions. Here, we summarize the advantages of solution-state (19)F NMR for studying the interactions of peptides and proteins with other biological molecules, review methods for the production of fluorine-labeled materials, and describe some representative recent examples in which (19)F NMR has been used to study conformational changes in peptides and proteins and their interactions with other biological molecules.

Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: Investigation of kinetics and requirement for an external electron donor

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen [Das et al. (2011) Angew. Chem. 50, 7148-7152]. Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions and that the oxygen in formate derives from water. The very low activity (<1 turnover/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turnover, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, k(prod) = 3.4 ± 0.5 min(-1), is observed, indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, k(cat) = 0.17 ± 0.01 min(-1) and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.

Noncovalent self-assembly of a heterotetrameric diiron protein

Diiron and dimanganese proteins catalyze a wide range of hydrolytic and oxygen-dependent reactions. To probe the mechanisms by which individual members of this class of proteins are able to catalyze such a wide range of reactions, we have prepared a model four-helix bundle with a diiron site located near the center of the bundle. The four-helix bundle is constructed by the noncovalent self-assembly of three different chains (Aa, Ab, and B) that self-assemble into the desired heterotetramer when mixed in a 1:1:2 molar ratio. On addition of ferrous ions and oxygen, the protein forms a complex with a UV-visible spectrum closely resembling that of peroxo-bridged diferric species in natural proteins and model compounds. By combining a small collection of n variants of these peptides, it should now be possible to prepare an n3 member library, which will allow systematic exploration of the features giving rise to the catalytic properties of this class of proteins.