Laminar flow-based microfluidic systems for molecular interaction analysis-Part 1: Chip development, system operation and measurement setup.

The recent advent of laminar flow-based microfluidic systems for molecular interaction analysis has enabled transformative new profiling of proteins in regards to their structure, disordering, complex formation and interactions in general. Based on the diffusive transport of molecules perpendicular to the direction of laminar flow in a microfluidic channel, systems of this type promise continuous-flow, high-throughput screening of complex, multi-molecule interactions, while remaining tolerant to heterogeneous mixtures. Using common microfluidic device processing, the technology provides unique opportunities, as well as device design and experimental challenges, for integrative sample handling approaches that can investigate biomolecular interaction events in complex samples with readily available laboratory equipment.

Laminar flow-based microfluidic systems for molecular interaction analysis-Part 2: Data extraction, processing and analysis.

The rate at which fluorescently-labeled biomolecules, that are flowing at a constant speed in a microfluidic channel, diffuse into an adjacent buffer stream can be used to calculate the diffusion coefficient of the molecule, which then gives a measure of its size. Experimentally, determining the rate of diffusion involves capturing concentration gradients in fluorescence microscopy images at different distances along the length of the microfluidic channel, where distance corresponds to residence time, based on the flow velocity. The preceding chapter in this journal covered the development of the experimental setup, including information about the microscope camera detection systems used to acquire fluorescence microscopy data.

On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications.

Within the complex milieu of a cell, which comprises a large number of different biomolecules, interactions are critical for function. In this post-reductionist era of biochemical research, the 'holy grail' for studying biomolecular interactions is to be able to characterize them in native environments. While there are a limited number of in situ experimental techniques currently available, there is a continuing need to develop new methods for the analysis of biomolecular complexes that can cope with the additional complexities introduced by native-like solutions.

Is now the time for a Rubiscuit or Ruburger? Increased interest in Rubisco as a food protein.

Much of the research on Rubisco aims at increasing crop yields, with the ultimate aim of increasing plant production to feed an increasing global population. However, since the identification of Rubisco as the most abundant protein in leaf material, it has also been touted as a direct source of dietary protein. The nutritional and functional properties of Rubisco are on a par with those of many animal proteins, and are superior to those of many other plant proteins.

An unexpected sticking point for carboxysome assembly.

In order to improve photosynthetic efficiency, bacteria often enclose RubisCO and carbonic anhydrase into microcompartments called carboxysomes. Assembly of these complexes requires a protein called CcmM. It had previously been thought that CcmM mediated RubisCO assembly by displacing one of the RubisCO subunits, Ryan show that despite having a three-dimensional structure that closely resembles the RubisCO small subunit, CcmM does not dislodge it, leading to a proposal for an alternative binding location.

Functional and solution structure studies of amino sugar deacetylase and deaminase enzymes from Staphylococcus aureus.

N-Acetylglucosamine-6-phosphate deacetylase (NagA) and glucosamine-6-phosphate deaminase (NagB) are branch point enzymes that direct amino sugars into different pathways. For Staphylococcus aureus NagA, analytical ultracentrifugation and small-angle X-ray scattering data demonstrate that it is an asymmetric dimer in solution. Initial rate experiments show hysteresis, which may be related to pathway regulation, and kinetic parameters similar to other bacterial isozymes.

A helical LC3-interacting region mediates the interaction between the retroviral restriction factor Trim5α and mammalian autophagy-related ATG8 proteins.

The retroviral restriction factor tripartite motif-containing 5α (Trim5α) acts during the early postentry stages of the retroviral life cycle to block infection by a broad range of retroviruses, disrupting reverse transcription and integration. The mechanism of this restriction is poorly understood, but it has recently been suggested to involve recruitment of components of the autophagy machinery, including members of the mammalian autophagy-related 8 (ATG8) family involved in targeting proteins to the autophagosome. To better understand the molecular details of this interaction, here we utilized analytical ultracentrifugation to characterize the binding of six ATG8 isoforms and determined the crystal structure of the Trim5α Bbox coiled-coil region in complex with one member of the mammalian ATG8 proteins, autophagy-related protein LC3 B (LC3B).

Self-assembly of toroidal proteins explored using native mass spectrometry.

The peroxiredoxins are a well characterised family of toroidal proteins which can self-assemble into a striking array of quaternary structures, including protein nanotubes, making them attractive as building blocks for nanotechnology. Tools to characterise these assemblies are currently scarce. Here, assemblies of peroxiredoxin proteins were examined using native mass spectrometry and complementary solution techniques.

Structural and functional analyses of Rubisco from arctic diatom species reveal unusual posttranslational modifications.

The catalytic performance of the major CO-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO sequestration in polar cold waters.

Immobilization of tobacco etch virus (TEV) protease on a high surface area protein nanofibril scaffold.

Tobacco etch virus (TEV) protease is widely used for the removal of poly-histidine affinity tags from proteins. In solution, it is a one-time use enzyme for tag cleavage that has low stability, and is therefore a good candidate for immobilization. Amyloid fibrils can act as a versatile nanoscaffold by providing a large surface area for biomolecule immobilization.

Multivalency regulates activity in an intrinsically disordered transcription factor.

The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy.

Quaternary structure influences the peroxidase activity of peroxiredoxin 3.

Peroxiredoxins are abundant peroxidase enzymes that are key regulators of the cellular redox environment. A major subgroup of these proteins, the typical 2-Cys peroxiredoxins, can switch between dimers and decameric or dodecameric rings, during the catalytic cycle. The necessity of this change in quaternary structure for function as a peroxidase is not fully understood.

Plant DHDPR forms a dimer with unique secondary structure features that preclude higher-order assembly.

Dihydrodipicolinate reductase (DHDPR) catalyses the second reaction in the diaminopimelate pathway of lysine biosynthesis in bacteria and plants. In contrast with the tetrameric bacterial DHDPR enzymes, we show that DHDPR from (grape) and are dimeric in solution. In the present study, we have also determined the crystal structures of DHDPR enzymes from the plants and , which are the first dimeric DHDPR structures.

Formation of supramolecular protein structures on gold surfaces.

Recent research has highlighted the exciting possibilities enabled by the use of protein structures as nanocomponents to form functional nanodevices. To this end, control over protein-protein and protein-surface interactions is essential. In this study, the authors probe the interaction of human peroxiredoxin 3 with gold surfaces, a protein that has been previously identified as having potential use in nanotechnology.

Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3.

Archaeal Lsm rings as stable self-assembling tectons for protein nanofabrication.

We have exploited the self-assembling properties of archaeal-derived protein Lsmα to generate new supramolecular forms based on its stable ring-shaped heptamer. We show that engineered ring tectons incorporating cysteine sidechains on obverse faces of the Lsmα toroid are capable of forming paired and stacked formations. A Cys-modified construct, N10C/E61C-Lsmα, appears to organize into disulfide-mediated tube formations up to 45 nm in length.

Dihydrodipicolinate Synthase: Structure, Dynamics, Function, and Evolution.

Enzymes are usually comprised of multiple subunits and more often than not they are made up of identical subunits. In this review we examine lysine biosynthesis and focus on the enzyme dihydrodipicolinate synthase in terms of its structure, function and the evolution of its varied number of subunits (quaternary structure). Dihydrodipicolinate synthase is the first committed step in the biosynthesis of lysine, which occurs naturally in plants, bacteria, archaea and fungi, but is not synthesized in mammals.

Structure and inhibition of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus.

N-Acetylneuraminate lyase is the first committed enzyme in the degradation of sialic acid by bacterial pathogens. In this study, we analyzed the kinetic parameters of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus (MRSA). We determined that the enzyme has a relatively high K of 3.

Measuring wildland fire fighter performance with wearable technology.

Wildland (rural) fire fighting is a physically demanding and hazardous occupation. An observational study was conducted to explore the use of new technologies for the field study of fire fighters at wildfires and to understand the work pressures of wildland fire fighting. The research was carried out with two fire fighters at real fires wearing microphones, miniature video cameras, heart rate monitors and GPS units to record their actions and location at wildfire events.

Structure and Function of Cyanobacterial DHDPS and DHDPR.

Lysine biosynthesis in bacteria and plants commences with a condensation reaction catalysed by dihydrodipicolinate synthase (DHDPS) followed by a reduction reaction catalysed by dihydrodipicolinate reductase (DHDPR). Interestingly, both DHDPS and DHDPR exist as different oligomeric forms in bacteria and plants. DHDPS is primarily a homotetramer in all species, but the architecture of the tetramer differs across kingdoms.

DNA G-quadruplexes show strong interaction with DNA methyltransferases in vitro.

The DNA methyltransferase enzymes (DNMTs) catalyzing cytosine methylation do so at specific locations of the genome, although with some level of redundancy. The de novo methyltransferases DNMT3A and 3B play a vital role in methylating the genome of the developing embryo in regions devoid of methylation marks. The ability of DNMTs to colocalize at sites of DNA damage is suggestive that recognition of mispaired bases and unusual structures is inherent to the function of these proteins.

The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain.

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus.

Peroxiredoxin is a versatile self-assembling tecton for protein nanotechnology.

The potential for protein tectons to be used in nanotechnology is increasingly recognized, but the repertoire of stable proteins that assemble into defined shapes in response to an environmental trigger is limited. Peroxiredoxins (Prxs) are a protein family that shows an amazing array of supramolecular assemblies, making them attractive tectons. Human Prx3 (hPrx3) forms toroidal oligomers characteristic of the Prx family, but no structure has been solved to date.

From knock-out phenotype to three-dimensional structure of a promising antibiotic target from Streptococcus pneumoniae.

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S.