A cheap and straightforward method for the selective isolation of histidine-derived natural products using nickel(II) phosphate.

Nickel-based resins have long been used in biochemical studies for the purification of His-tagged proteins, but their potential in the isolation of histidine-derived small molecules has not been investigated to date. Many agriculturally-important mycotoxins incorporate histidine residues, as do natural products from both plants and bacteria. Here, a highly-selective solid-phase extraction method is described for the purification of histidine-derived natural products using the insoluble nickel salt Ni(PO).

Structures of PKA-phospholamban complexes reveal a mechanism of familial dilated cardiomyopathy.

Several mutations identified in phospholamban (PLN) have been linked to familial dilated cardiomyopathy (DCM) and heart failure, yet the underlying molecular mechanism remains controversial. PLN interacts with sarco/endoplasmic reticulum Ca-ATPase (SERCA) and regulates calcium uptake, which is modulated by the protein kinase A (PKA)-dependent phosphorylation of PLN during the fight-or-flight response. Here, we present the crystal structures of the catalytic domain of mouse PKA in complex with wild-type and DCM-mutant PLNs.

Guest Sequence Can Influence RNA Encapsulation by an Engineered Cationic Protein Capsid.

Specific encapsulation of RNA in a cell is a challenging molecular recognition problem that has important implications for virology and drug delivery. An engineered variant of the lumazine synthase capsid that possesses a positively supercharged interior (AaLS-pos) has previously been shown to encapsulate a mixture of cellular RNAs in bacteria via charge complementarity. To investigate the influence of nucleotide sequence on encapsulation, eight reporter RNAs with the same charge but with highly diverse arbitrary sequence regions (ASRs) were coproduced with AaLS-pos in cells.

A Protein-Capsid-Based System for Cell Delivery of Selenocysteine.

Selenocysteine (Sec) has received a lot of attention as a potential anticancer drug. However, its broad cytotoxicity limits its therapeutic usefulness. Thus, Sec is an attractive candidate for targeted drug delivery.

Encapsulation and Controlled Release of Protein Guests by the Bacillus subtilis Lumazine Synthase Capsid.

In Bacillus subtilis, the 60-subunit dodecahedral capsid formed by lumazine synthase (BsLS) acts as a container for trimeric riboflavin synthase (BsRS). To test whether the C-terminal sequence of BsRS is responsible for its encapsulation by BsLS, the green fluorescent protein (GFP) was fused to either the last 11 or the last 32 amino acids of BsRS, yielding variant GFP11 or GFP32, respectively. After purification, BsLS capsids that had been co-produced in bacteria with GFP11 and GFP32 are 15- and 6-fold more fluorescent, respectively, than BsLS co-produced with GFP lacking any BsRS fragment, indicating complex formation.

In vivo encapsulation of nucleic acids using an engineered nonviral protein capsid.

In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems.

Conversion of a dodecahedral protein capsid into pentamers via minimal point mutations.

Protein self-assembly relies upon the formation of stabilizing noncovalent interactions across subunit interfaces. Identifying the determinants of self-assembly is crucial for understanding structure-function relationships in symmetric protein complexes and for engineering responsive nanoscale architectures for applications in medicine and biotechnology. Lumazine synthases (LS's) comprise a protein family that forms diverse quaternary structures, including pentamers and 60-subunit dodecahedral capsids.

Reagentless oxidative folding of disulfide-rich peptides catalyzed by an intramolecular diselenide.

In cysteine-rich peptides, diselenides can be used as a proxy for disulfide bridges, since the energetic preference for diselenide bonding over mixed selenium-sulfur bonds simplifies folding. Herein we report that an intramolecular diselenide bond efficiently catalyzes the oxidative folding of selenopeptide analogs of conotoxins, and serves as a reagentless method to substantially accelerate formation of various native disulfide bridging patterns.

Directed evolution of a protein container.

Confinement of enzymes in protein nanocompartments represents a potentially powerful strategy for controlling catalytic activity in cells. By using a simple electrostatically based tagging system for protein encapsulation, we successfully sequestered HIV protease, a toxic enzyme when produced cytoplasmically, within an engineered lumazine synthase capsid. The growth advantage resulting from protecting the Escherichia coli host from the protease enabled directed evolution of improved capsids.

Diselenides as universal oxidative folding catalysts of diverse proteins.

Small-molecule diselenides show considerable potential as catalysts of oxidative protein folding. To explore their scope, diselenide-containing redox buffers were used to promote the folding of proteins that varied in properties such as size, overall tertiary structure, number of disulfide bonds, pI value, and difficulty of in vitro folding. Diselenides are able to catalyze the oxidative folding of all proteins tested, providing significant increases in both rate and yield relative to analogous disulfides.

Small-molecule diselenides catalyze oxidative protein folding in vivo.

Prokaryotic cells normally rely on periplasmic oxidoreductases to promote oxidative protein folding. Here we show that simple diselenides can also facilitate the conversion of dithiols to disulfides in vivo, functionally replacing one such oxidoreductase, DsbA, in the oxidative folding of diverse proteins. Structurally analogous disulfides provide no detectable effect when used at concentrations that gave optimal activity with diselenides, and even at 100- to 1000-fold higher levels they show only partial activity.

Relative tolerance of an enzymatic molten globule and its thermostable counterpart to point mutation.

Enzyme structures reflect the complex interplay between the free energy of unfolding (DeltaG) and catalytic efficiency. Consequently, the effects of point mutations on structure, stability, and function are difficult to predict. It has been proposed that the mutational robustness of homologous enzymes correlates with a higher initial DeltaG.

Catalysis of oxidative protein folding by small-molecule diselenides.

The production of recombinant, disulfide-containing proteins often requires oxidative folding in vitro. Here, we show that diselenides, such as selenoglutathione, catalyze oxidative protein folding by O 2. Substantially lower concentrations of a redox buffer composed of selenoglutathione and the thiol form of glutathione can consequently be used to achieve the same rate and yield of folding as a standard glutathione redox buffer.

Minimalist active-site redesign: teaching old enzymes new tricks.

Although nature evolves its catalysts over millions of years, enzyme engineers try to do it a bit faster. Enzyme active sites provide highly optimized microenvironments for the catalysis of biologically useful chemical transformations. Consequently, changes at these centers can have large effects on enzyme activity.

Selenoglutathione: efficient oxidative protein folding by a diselenide.

Diselenide bonds are intrinsically more stable than disulfide bonds. To examine how this stability difference affects reactivity, we synthesized selenoglutathione (GSeSeG), an analogue of the oxidized form of the tripeptide glutathione that contains a diselenide bond in place of the natural disulfide. The reduction potential of this diselenide bond was determined to be -407 +/- 9 mV, a value which is 151 mV lower than that of the disulfide bond in glutathione (GSSG).

Novel enzymes through design and evolution.

The generation of enzymes with new catalytic activities remains a major challenge. So far, several different strategies have been developed to tackle this problem, including site-directed mutagenesis, random mutagenesis (directed evolution), antibody catalysis, computational redesign, and de novo methods. Using these techniques, a broad array of novel enzymes has been created (aldolases, decarboxylases, dehydratases, isomerases, oxidases, reductases, and others), although their low efficiencies (10 to 100 M(-1) s(-l)) compared to those of the best natural enzymes (10(6) to 10(8) M(-1) s(-1)) remains a significant concern.

Tunnel plasticity and quaternary structural integrity of a pentameric protein ring.

Cyclic protein oligomers are common in cells. However, the importance of the residues that line the central tunnel of protein rings for overall architectural integrity is not well understood. To investigate the role of tunnel positions in protein assembly and stability, we prepared variants of the homo-pentameric lumazine synthase (LS) from Saccharomyces cerevisiae in which the three residues that line the middle of the tunnel were simultaneously changed.

A simple tagging system for protein encapsulation.

Molecular containers that encapsulate specific cargo can be useful for many natural and non-natural processes. We report a simple system, based on charge complementarity, for the encapsulation of appropriately tagged proteins within an engineered, proteinaceous capsid. Four negative charges per monomer were added to the lumazine synthase from Aquifex aeolicus (AaLS).

Recombination of fragmented proteins.

Directed evolution is a powerful method for generating novel molecules with desirable properties. In developing a new sensor to screen for protein-protein interactions, Tafelmeyer et al. report a clever strategy to evolve heterodimeric "split proteins" from a monomer in this issue of Chemistry & Biology.

Deciphering enzymes. Genetic selection as a probe of structure and mechanism.

The efficient engineering of enzymes with novel activities remains an ongoing challenge. Towards this end, genetic selection techniques provide a method for finding rare solutions to catalytic problems that requires only a limited foreknowledge of structure-function relationships. We have used genetic selections to extensively probe the structure and mechanism of chorismate mutases.

Catalysis of protein folding by an immobilized small-molecule dithiol.

The isomerization of non-native disulfide bonds often limits the rate of protein folding. Small-molecule dithiols can catalyze this process. Here, a symmetric trithiol, tris(2-mercaptoacetamidoethyl)amine, is designed on the basis of criteria known to be important for efficient catalysis of oxidative protein folding.

The CXC motif: a functional mimic of protein disulfide isomerase.

Protein disulfide isomerase (PDI) utilizes the active site sequence Cys-Gly-His-Cys (CGHC; E degrees ' = -180 mV) to effect thiol-disulfide interchange during oxidative protein folding. Here, the Cys-Gly-Cys-NH(2) (CGC) peptide is shown to have a disulfide reduction potential (E degrees ' = -167 mV) that is close to that of PDI. This peptide has a thiol acid dissociation constant (pK(a) = 8.