A Suite of Engineered GFP Molecules for Oligomeric Scaffolding.

Applications ranging from synthetic biology to protein crystallization could be advanced by facile systems for connecting multiple proteins together in predefined spatial relationships. One approach to this goal is to engineer many distinct assembly forms of a single carrier protein or scaffold, to which other proteins of interest can then be readily attached. In this work we chose GFP as a scaffold and engineered many alternative oligomeric forms, driven by either specific disulfide bond formation or metal ion addition.

An allosteric model for control of pore opening by substrate binding in the EutL microcompartment shell protein.

The ethanolamine utilization (Eut) microcompartment is a protein-based metabolic organelle that is strongly associated with pathogenesis in bacteria that inhabit the human gut. The exterior shell of this elaborate protein complex is composed from a few thousand copies of BMC-domain shell proteins, which form a semi-permeable diffusion barrier that provides the interior enzymes with substrates and cofactors while simultaneously retaining metabolic intermediates. The ability of this protein shell to regulate passage of substrate and cofactor molecules is critical for microcompartment function, but the details of how this diffusion barrier can allow the passage of large cofactors while still retaining small intermediates remain unclear.

Transmission of malaria to mosquitoes blocked by bumped kinase inhibitors.

Effective control and eradication of malaria will require new tools to prevent transmission. Current antimalarial therapies targeting the asexual stage of Plasmodium do not prevent transmission of circulating gametocytes from infected humans to mosquitoes. Here, we describe a new class of transmission-blocking compounds, bumped kinase inhibitors (BKIs), which inhibit microgametocyte exflagellation.

Multiple determinants for selective inhibition of apicomplexan calcium-dependent protein kinase CDPK1.

Diseases caused by the apicomplexan protozoans Toxoplasma gondii and Cryptosporidium parvum are a major health concern. The life cycle of these parasites is regulated by a family of calcium-dependent protein kinases (CDPKs) that have no direct homologues in the human host. Fortuitously, CDPK1 from both parasites contains a rare glycine gatekeeper residue adjacent to the ATP-binding pocket.

Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis.

Background: Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis.

Results: Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis.

Structure of the cystathionine γ-synthase MetB from Mycobacterium ulcerans.

Cystathionine γ-synthase (CGS) is a transulfurication enzyme that catalyzes the first specific step in L-methionine biosynthesis by the reaction of O(4)-succinyl-L-homoserine and L-cysteine to produce L-cystathionine and succinate. Controlling the first step in L-methionine biosythesis, CGS is an excellent potential drug target. Mycobacterium ulcerans is a slow-growing mycobacterium that is the third most common form of mycobacterial infection, mainly infecting people in Africa, Australia and Southeast Asia.

Structure of thymidylate kinase from Ehrlichia chaffeensis.

The enzyme thymidylate kinase phosphorylates the substrate thymidine 5'-phosphate (dTMP) to form thymidine 5'-diphosphate (dTDP), which is further phosphorylated to dTTP for incorporation into DNA. Ehrlichia chaffeensis is the etiologic agent of human monocytotropic erlichiosis (HME), a potentially life-threatening tick-borne infection. HME is endemic in the United States from the southern states up to the eastern seaboard.

Structure of a cyclin-dependent kinase from Giardia lamblia.

Giardia lamblia is the etiologic agent of giardiasis, a water-borne infection that is prevalent throughout the world. The need for new therapeutics for the treatment of giardiasis is of paramount importance. Owing to the ubiquitous nature of kinases and their vital importance in organisms, they are potential drug targets.

Structures of a putative ζ-class glutathione S-transferase from the pathogenic fungus Coccidioides immitis.

Coccidioides immitis is a pathogenic fungus populating the southwestern United States and is a causative agent of coccidioidomycosis, sometimes referred to as Valley Fever. Although the genome of this fungus has been sequenced, many operons are not properly annotated. Crystal structures are presented for a putative uncharacterized protein that shares sequence similarity with ζ-class glutathione S-transferases (GSTs) in both apo and glutathione-bound forms.

Structures of phosphopantetheine adenylyltransferase from Burkholderia pseudomallei.

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the fourth of five steps in the coenzyme A biosynthetic pathway, reversibly transferring an adenylyl group from ATP onto 4'-phosphopantetheine to yield dephospho-coenzyme A and pyrophosphate. Burkholderia pseudomallei is a soil- and water-borne pathogenic bacterium and the etiologic agent of melioidosis, a potentially fatal systemic disease present in southeast Asia. Two crystal structures are presented of the PPAT from B.

Identification of inhibitors for putative malaria drug targets among novel antimalarial compounds.

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of P.

Use of thermal melt curves to assess the quality of enzyme preparations.

This study sought to determine whether the quality of enzyme preparations can be determined from their melting curves, which may easily be obtained using a fluorescent probe and a standard reverse transcription-polymerase chain reaction (RT-PCR) machine. Thermal melt data on 31 recombinant enzymes from Plasmodium parasites were acquired by incrementally heating them to 90 degrees C and measuring unfolding with a fluorescent dye. Activity assays specific to each enzyme were also performed.

Buffer optimization of thermal melt assays of Plasmodium proteins for detection of small-molecule ligands.

In the past decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential ligands.