Revisit the effect of fibrillization on functions of prion protein from the perspective of Cu(II) binding.

Conversion of prion protein (PrP) from its α-helical form to a β-sheet rich scrapie form constitutes the key event of the etiology of prion diseases. Fundamental questions remain concerning the functions of prion protein and the mechanisms leading to the formation of misfolded forms. A wealth of evidence links physiological functions of PrP to its ability to bind Cu(II), suggesting that it may act as a copper buffer or be part of the copper transportation system.

The evolution of RuBisCO stability at the thermal limit of photoautotrophy.

A long-standing question in evolutionary biology is how organisms adapt to novel environments. In North American hot springs, diversification of a clade of the cyanobacterium Synechococcus into hotter environments has resulted in the unique innovation of a light-driven ecosystem at temperatures up to 74°C, and temperature adaptation of photosynthetic carbon fixation with the Calvin cycle contributed to this process. Here, we investigated the evolution of thermostability of the Calvin cycle enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) during Synechococcus divergence.

Dissociation of recombinant prion protein fibrils into short protofilaments: implications for the endocytic pathway and involvement of the N-terminal domain.

Fibril dissociation is necessary for efficient conversion of normal prion protein to its misfolded state and continued propagation into amyloid. Recent studies have revealed that conversion occurs along the endocytic pathway. To improve our understanding of the dissociation process, we have investigated the effect of low pH on the stability of recombinant prion fibrils.

Use of microcalorimetry to determine the costs and benefits to Pseudomonas putida strain KT2440 of harboring cadmium efflux genes.

A novel microcalorimetric approach was used to analyze the responses of a metal-tolerant soil bacterium (Pseudomonas putida strain KT2440) to metal resistance gene deletions in cadmium-amended media. As hypothesized, under cadmium stress, the wild-type strain benefited from the resistance genes by entering the exponential growth phase earlier than two knockout strains. In the absence of cadmium, strain KT1, carrying a deletion in the main component (czcA1) of a Cd/Zn chemiosmotic efflux transporter (CzcCBA1), grew more efficiently than the wild type and released ∼700 kJ (per mole of biomass carbon) less heat than the wild-type strain, showing the energetic cost of maintaining CzcCBA1 in the absence of cadmium.

Alternating pattern of stereochemistry in the nonactin macrocycle is required for antibacterial activity and efficient ion binding.

Nonactin is a polyketide antibiotic produced by Streptomyces griseus ETH A7796 and is an ionophore that is selective for K(+) ions. It is a cyclic tetraester generated from two monomers of (+)-nonactic acid and two of (-)-nonactic acid, arranged (+)-(-)-(+)-(-) so that nonactin has S4 symmetry and is achiral. To understand why achiral nonactin is the naturally generated diastereoisomer, we generated two alternate diastereoisomers of nonactin, one prepared solely from (+)-nonactic acid and one prepared solely from (-)-nonactic acid, referred to here as 'all-(+)-nonactin' and 'all-(-)-nonactin', respectively.

Hydroxylase activity of Met471Cys tyramine beta-monooxygenase.

A series of mutations was targeted at the methionine residue, Met471, coordinating the Cu(M) site of tyramine beta-monooxygenase (TbetaM). The methionine ligand at Cu(M) is believed to be key to dioxygen activation and the hydroxylation chemistry of the copper monooxygenases. The reactivity and copper binding properties of three TbetaM mutants, Met471Asp, Met471Cys, and Met471His, were examined.

Mechanism of the insect enzyme, tyramine beta-monooxygenase, reveals differences from the mammalian enzyme, dopamine beta-monooxygenase.

Tyramine beta-monooxygenase (TbetaM) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of TbetaM for comparison with the homologous mammalian enzymes, dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase. A new and distinctive feature of TbetaM is very strong substrate inhibition that is dependent on the level of the co-substrate, O(2), and reductant as well as substrate deuteration.

Probing structural differences in prion protein isoforms by tyrosine nitration.

Two conformational isomers of recombinant hamster prion protein (residues 90-232) have been probed by reaction with two tyrosine nitration reagents, peroxynitrite and tetranitromethane. Two conserved tyrosine residues (tyrosines 149 and 150) are not labeled by either reagent in the normal cellular form of the prion protein. These residues become reactive after the protein has been converted to the beta-oligomeric isoform, which is used as a model of the fibrillar form that causes disease.

Modulation of the electrochemical behavior of tyrosyl radicals by the electrode surface.

The ability to adsorb proteins and enzymes on electrode surfaces enhances opportunities for studying enzyme activity and redox-based catalysis. Proteins may be bound in a chosen orientation on surfaces so that specific sites within them may be preferentially studied, but to date no systematic study of a redox moiety from solvent to electrode surface to the protein milieu has been performed. We report the redox and ionization behavior of tyrosine-cysteine, using the cysteine residue to form covalent linkages with Au and self-assembled-monolayer (SAM)-modified Au surfaces and using the tyrosine for redox activity.

Structural studies of Apo NosL, an accessory protein of the nitrous oxide reductase system: insights from structural homology with MerB, a mercury resistance protein.

The formation of the unique catalytic tetranuclear copper cluster (Cu(Z)) of nitrous oxide reductase, N(2)OR, requires the coexpression of a multiprotein assembly apparatus encoded by the nosDFYL operon. NosL, one of the proteins encoded by this transcript, is a 20 kDa lipoprotein of the periplasm that has been shown to bind copper(I), although its function has yet to be detemined. Cu(I) EXAFS data collected on the holo protein demonstrated that features of the copper binding site are consistent with a role for this protein as a metallochaperone, a class of metal ion transporters involved in metal resistance, homeostasis, and metallocluster biosynthesis.

Expression and characterization of recombinant tyramine beta-monooxygenase from Drosophila: a monomeric copper-containing hydroxylase.

We report here the development of a robust recombinant expression system for Drosophila melanogaster tyramine beta-monooxygenase (TbetaM), the insect analog of mammalian dopamine beta-monooxygenase. Recombinant TbetaM is rapidly purified from the host cell media in three chromatographic steps. The expression system produces approximately 3-10 mg of highly purified, active protein per liter of culture.

Cloning, heterologous expression, and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris.

The cytochrome (cyt) c', cyt c(556), and cyt c(2) genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c' and cyt c(556) have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b(562), in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues.