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. Highresolution 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.

Adenosylcobalamin-dependent glutamate mutase catalyzes an unusual carbon skeleton rearrangement that proceeds through the formation of free radical intermediates generated by the substrate-induced cleavage of the coenzyme cobalt-carbon bond. The reaction was studied at 10 degrees C with various concentrations of L-glutamate and L-threo-3-methylaspartate and with use of stopped-flow spectroscopy to follow the formation of cob(II)alamin. Either substrate induces rapid formation of cob(II)alamin, which accumulates to account for about 25% of the total enzyme species in the steady state when substrate is saturating. Measurements of the rate constant for the formation of cob(II)alamin demonstrate that the enzyme accelerates the rate of homolysis of the cobalt-carbon bond by at least 10(12)-fold. Very large isotope effects on cob(II)alamin formation, of 28 and 35, are observed with deuterated L-glutamate and deuterated L-threo-3-methylaspartate, respectively. This implies a mechanism in which Co-C bond homolysis is kinetically coupled to substrate hydrogen abstraction. Therefore, adenosyl radical can only be formed as a high-energy intermediate only at very low concentrations on the enzyme. The magnitude of the isotope effects suggests that hydrogen tunneling may play an important role catalysis.

Just add water: Structurally, cyanobacterial aldehyde decarbonylases are members of the non‐heme diiron oxygenase family of enzymes. However, the enzyme catalyzes the hydrolysis of aliphatic aldehydes to alkanes and formate (see scheme), in an oxygen‐independent reaction. This unusual and chemically difficult reaction most likely involves free radical intermediates.

ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 A Ê ) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O 2 /H 2 O gate with a putative protein channel. This is the ®rst crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme±substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure.

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.

Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (DeltaDeltaG(unfold)) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with L-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). We find that DeltaDeltaG(unfold) is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in DeltaDeltaG(unfold) of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally from partitioning measurements on hFLeu and leucine. The increased stability of this fluorinated protein may therefore be ascribed to simple hydrophobic effects, rather than specific "fluorous" interactions between the hFLeu residues.

The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C 3 -symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C 4 -symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C 4 and C 3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.coiled coils | protein design | native mass spectrometry | analytical ultracentrifugation | cryoelectron microscopy

In the commonly used nucleation-dependent model of protein aggregation, aggregation proceeds only after a lag phase in which the concentration of energetically unfavorable nuclei reaches a critical value. The formation of oligomeric species prior to aggregation can be difficult to detect by current spectroscopic techniques. By using real-time 19F NMR along with other techniques, we are able to show that multiple oligomeric species can be detected during the lag phase of Aβ1-40 fiber formation, consistent with a complex mechanism of aggregation. At least 6 types of oligomers can be detected by 19F NMR. These include the reversible formation of large β-sheet oligomer immediately after solubilization at high peptide concentration; a small oligomer that forms transiently during the early stages of the lag phase; and 4 spectroscopically distinct forms of oligomers with molecular weights between ~30–100 kDa that appear during the later stages of aggregation. The ability to resolve individual oligomers and track their formation in real-time should prove fruitful in understanding the aggregation of amyloidogenic proteins and in isolating potentially toxic non-amyloid oligomers.