Observation of a single protein by ultrafast X-ray diffraction.

The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes.

Graphene-based phenformin carriers for cancer cell treatment: a comparative study between oxidized and pegylated pristine graphene in human cells and zebrafish.

Graphene is an attractive choice for the development of an effective drug carrier in cancer treatment due to its high adsorption area and pH-responsive drug affinity. In combination with the highly potent metabolic drug phenformin, increased doses could be efficiently delivered to cancer cells. This study compares the use of graphene oxide (GO) and polyethylene glycol stabilized (PEGylated) pristine graphene nanosheets (PGNSs) for drug delivery applications with phenformin.

Improved pH-Responsive Release of Phenformin from Low-Defect Graphene Compared to Graphene Oxide.

Graphene-based drug carriers provide a promising addition to current cancer drug delivery options. Increased accessibility of high-quality graphene made by plasma-enhanced chemical vapor deposition (PE-CVD) makes it an attractive material to revisit in comparison to the widely studied graphene oxide (GO) in drug delivery. Here, we show the potential of repurposing the metabolic drug phenformin for cancer treatment in terms of stability, binding, and pH-responsive release.

Characterisation of a novel cold-adapted calcium-activated transglutaminase: implications for medicine and food processing.

Transglutaminases are a family of enzymes that catalyse the cross-linking of proteins by forming covalent bonds between lysine and glutamine residues in various polypeptides. Cross-linking reactions are involved in blood clots, skin formation, embryogenesis and apoptosis. Clinically, these enzymes appear to be implicated in neurodegenerative diseases, tumours and coeliac diseases.

'Birth defects' of photosystem II make it highly susceptible to photodamage during chloroplast biogenesis.

High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water-oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodeling.

A strategy to characterize chlorophyll protein interaction in LIL3.

Background: The function of proteins is at large determined by cofactors selectively bound to protein structure. Without chlorophyll specifically bound to protein, light harvesting and photosynthesis would not be possible. The binding of chlorophyll to light harvesting proteins has been extensively studied in reconstitution assays using proteins expressed in vitro; however, the mechanism of the reconstitution reaction remained unclear.

Characterization of chlorophyll binding to LIL3.

The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles.

Supercomplexes of plant photosystem I with cytochrome b6f, light-harvesting complex II and NDH.

Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome bf (Cytbf) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking.

Biogenesis of water splitting by photosystem II during de-etiolation of barley (Hordeum vulgare L.).

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de-etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development.

Lil3 dimerization and chlorophyll binding in Arabidopsis thaliana.

The two-helix light harvesting like (Lil) protein Lil3 belongs to the family of chlorophyll binding light harvesting proteins of photosynthetic membranes. A function in tetrapyrrol synthesis and stabilization of geranylgeraniol reductase has been shown. Lil proteins contain the chlorophyll a/b-binding motif; however, binding of chlorophyll has not been demonstrated.

Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley.

The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3.

Free flow electrophoresis for separation of native membrane protein complexes.

This chapter describes the technology of free flow electrophoresis (FFE) and protocols to separate membrane protein complexes for proteome analysis. FFE is a highly versatile technology applied in the field of protein analysis. It is superior to native PAGE due to its fast continuous processing of sample at high resolution.

Down-regulation of ZmEXPB6 (Zea mays β-expansin 6) protein is correlated with salt-mediated growth reduction in the leaves of Z. mays L.

The salt-sensitive crop Zea mays L. shows a rapid leaf growth reduction upon NaCl stress. There is increasing evidence that salinity impairs the ability of the cell walls to expand, ultimately inhibiting growth.

Separation of membrane protein complexes by native LDS-PAGE.

Gel electrophoresis has become one of the most important methods for the analysis of proteins and protein complexes in a molecular weight range of 1-10(7) kDa. The separation of membrane protein complexes remained challenging to standardize until the demonstration of Blue Native PAGE in 1991 [1] and Clear Native PAGE in 1994 [2]. We present a robust protocol for high-resolution separation of photosynthetic complexes from Arabidopsis thaliana using lithium dodecyl sulfate as anion in a modified Blue Native PAGE (LDS-PAGE).

Analysis of the chloroplast proteome in arc mutants and identification of novel protein components associated with FtsZ2.

Chloroplasts are descendants of cyanobacteria and divide by binary fission. The number of chloroplasts is regulated in a cell type-specific manner to ensure that specialized cell types can perform their functions optimally. Several protein components of the chloroplast division apparatus have been identified in the past several years, but how this process is regulated in response to developmental status, environmental signals and stress is still unknown.

Native DIGE of fluorescent plant protein complexes.

CyDye labeling and DIGE have not only been proven to work for soluble proteins but also at the level of whole membrane protein complexes. After complex solubilization and CyDye labeling, proteins can be separated by native PAGE which is often combined with SDS PAGE in a subsequent step. By this combination, sizes of complexes as well as their subunit composition can be compared after mixing samples from different physiological states.

Proteomic comparison of etioplast and chloroplast protein complexes.

Angiosperms grown in darkness develop etioplasts during skotomorphogenesis. It is well known that etioplasts accumulate large quantities of protochlorophyllideoxidoreductase, are devoid of chlorophyll and are the site to assemble the photosynthetic machinery during photomorphogenesis. Proteomic investigation of the membrane protein complexes by Native PAGE, in combination with CyDye labelling and mass spectrometric analysis revealed that etioplasts and chloroplasts share a number of membrane protein complexes characteristic for electron transport, chlorophyll and protein synthesis as well as fatty acid biosynthesis.

Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes.

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first.

Organelle proteomics: reduction of sample complexity by enzymatic in-gel selection of native proteins.

One major problem in proteomics is the biochemical complexity of living cells. Therefore, strategies are needed to reduce the number of proteins to a manageable amount, enabling researchers to make a statement concerning protein functions. One possibility is the isolation of organelles, which reduces the protein complexity, e.

Organelle proteomics.

The proteome of the cell is at the frontier of being too complex for proteomic analysis. Organelles provide a step up. Organelles compartmentalize the cell enabling a proteome, physiology and metabolism analysis in time and in space.

Identification of the N-termini of NADPH : protochlorophyllide oxidoreductase A and B from barley etioplasts (Hordeum vulgare L.).

The N-termini of the NADPH : protochlorophyllide oxidoreductase (POR) proteins A and B from barley and POR from pea were determined by acetylation of the proteins and selective isolation of the N-terminal peptides for mass spectrometry de novo sequence analysis. We show that the cleavage sites between the transit peptides and the three mature POR proteins are homologous. The N-terminus in PORA is V48, that in PORB is A61, and that in POR from pea is E64.

Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts.

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN-PAGE separation of membrane protein complexes and selective MS that the accumulation of one-helix proteins from PSII is light independent and occurs in etioplasts.

Impact of PsbTc on forward and back electron flow, assembly, and phosphorylation patterns of photosystem II in tobacco.

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn(4)Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth.

Localization of 13 one-helix integral membrane proteins in photosystem II subcomplexes.

Photosystem II is a multimeric protein complex of the thylakoid membrane in chloroplasts. Approximately half of the at least 26 different integral membrane protein subunits have molecular masses lower than 10 kDa. After one-dimensional (1D) or two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) separation, followed by enzymatic digestion of detected proteins, hardly any of these low-molecular-weight (LMW) subunits are detectable.