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. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.

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. The cytotoxicity and mitotoxicity of the graphene-based systems were assessed in human cells and zebrafish larvae. Targeted drug release from GO and PGNSs was evaluated at different pH levels known to arise in proliferating tumor microenvironments. PGNSs were less cytotoxic and mitotoxic than GO, and showed an increased release of phenformin at lower pH in cells, compared to GO. In addition, the systemic phenformin effect was mitigated in zebrafish larvae when bound to GO and PGNSs compared to free phenformin, as measured by flavin metabolic lifetime imaging. These results pave the way for improved phenformin-based cancer therapy using graphene nano-sheets, where PGNSs were superior to GO.

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. Using covalent attachment of poly(ethylene glycol) (PEG) onto pristine (PE-CVD) graphene, we show that PEG stabilized graphene nanosheets (PGNS) are stable in aqueous solutions and exhibit higher binding affinity toward phenformin than GO. Moreover, we experimentally demonstrate an improved drug release from PGNS than GO at pH levels lower than physiological conditions, yet comparable to that found in tumor microenvironments.

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. Transglutaminases have great potential for use in the food industry because of their ability to cross-link proteins that are not normally linked. Here, a gene coding for transglutaminase from Atlantic cod was cloned into a bacterial expression vector and used to transform protein expression in a strain of Escherichia coli. The successful expression of recombinant transglutaminase protein from Atlantic cod (AcTG-1) as a soluble protein upon induction at low temperature was confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry analysis. Biochemical characterisation demonstrated that the transglutaminase was active between 0 and 65 °C, but was completely inactivated after 20-min incubation at 70 °C. Interestingly, the enzyme displayed cold-adapted features, such as temperature instability combined with high catalytic efficiency at low temperatures (8-16 °C). In addition, the enzyme had optimal activity at 50 °C, a new feature for a cold-adapted enzyme. AcTG-1 was active in the pH range from 6 to 9, with an optimum at pH 8, and required 5 mm calcium for maximum activity. Potential calcium-binding sites in the enzyme were predictable, making the enzyme an appropriate model for studying structure-function relationships in the calcium-dependent transglutaminase family. In vitro gel analysis revealed that transglutaminase cross-linked casein, collagen and gelatin. The binding of fish fillets in the presence of recombinant AcTG-1 provided further macroscopic proof for the potential application of AcTG-1 as a biological cross-linker in the food industry. Once binding occurred, fish fillets withstood further processing such as frying, boiling, freeze-thawing and chilling. The low-temperature activity and new enzymatic properties of AcTG-1 appear to offer advantages over commercially available enzymatic glues in the food industry.

'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. Employing membrane-inlet mass spectrometry and O2 -polarography under flashing light conditions, we demonstrate that newly synthesized PSII complexes are far more susceptible to photodamage than are mature PSII complexes. We examined these 'PSII birth defects' in barley seedlings and plastids (etiochloroplasts and chloroplasts) isolated at various times during de-etiolation as chloroplast development begins and matures in synchronization with thylakoid membrane biogenesis and grana membrane formation. We show that the degree of PSII photodamage decreases simultaneously with biogenesis of the PSII turnover efficiency measured by O2 -polarography, and with grana membrane stacking, as determined by electron microscopy. Our data from fluorescence, QB -inhibitor binding, and thermoluminescence studies indicate that the decline of the high-light susceptibility of PSII to photodamage is coincident with appearance of electron transfer capability QA - → QB during de-etiolation. This rate depends in turn on the downstream clearing of electrons upon buildup of the complete linear electron transfer chain and the formation of stacked grana membranes capable of longer-range energy transfer.

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. We have shown that membrane integral light-harvesting-like protein, LIL3, binds chlorophyll a with a Kd of 146 nM in vitro by thermophoresis. Here, reconstitution of chlorophyll binding to LIL3 has been characterized by four different methods. RESULTS: Structural changes in the reconstitution process have been investigated by light-scattering and differential Trp-fluorescence. For characterization of the chlorophyll binding site at LIL3, the analysis of LIL3 mutants has been conducted using native PAGE and thermophoresis. We find that the oxidized state of dithiothreitol is the essential component for reconstitution of chlorophyll binding to LIL3 in n-Dodecyl ß-d-maltoside micelles at RT. Chlorophyll increased the polydispersity of the micellar states while dithiothreitol maintained LIL3 in a partially unfolded state at RT. Dimerization of LIL3 was abolished if amino acids N174, R176, and E171 were mutated to Ala; while, chlorophyll binding to LIL3 was abolished in mutant N174A, but retained in E171A, and R176A albeit at an about six- and five-fold decreased dissociation constant. Results show that N174 of LIL3 is essential for binding chlorophyll a. CONCLUSIONS: Chlorophyll binding to LIL3 can be shown by thermophoresis, and native gel electrophoresis, while analysis of reconstitution conditions by dynamic light scattering and differential scanning fluorometry are of critical importance for method optimization.

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. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3.

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 b6f (Cytb6f) 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. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb6f at the antenna side of PSI. The rectangular-shaped Cytb6f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1-3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb6f, may improve regulation of electron transport by the control of binding partners and distances in small domains.

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. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll-binding protein complexes and development of water splitting via O2 production in plastids (etiochloroplasts) isolated during de-etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light-harvesting antenna [PSII-light-harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de-etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de-etiolation, etiochloroplasts revealed the same water-splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de-etiolation precedes assembly of the PSII-LHCII supercomplexes. Taken together, data show a rapid establishment of water-splitting activity during etioplast-to-chloroplast transition and emphasize that assembly of the functional water-splitting site of PSII is not the rate-limiting step in the formation of photoactive thylakoid membranes.

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. We find that Lil3.2 from Arabidopsis thaliana forms heterodimers with Lil3.1 and binds chlorophyll. Lil3.2 heterodimerization (25±7.8 nM) is favored relative to homodimerization (431±59 nM). Interaction of Lil3.2 with chlorophyll a (231±49 nM) suggests that heterodimerization precedes binding of chlorophyll in Arabidopsis thaliana.

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. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).

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. Additionally, the dynamic separation range from ions, peptides, to proteins, protein complexes, up to organelles, and whole cells makes it the method of choice in the analysis of proteins. FFE is carried out in an aqueous medium without inducing any solid matrix, such as acrylamide, so that it simplifies the analysis of protein complexes for the downstream analysis. Here, we describe the novel zone electrophoresis interval method (IZE-FFE) for separation of protein complexes from the thylakoid membrane of Arabidopsis thaliana by charge only. Protein complexes isolated by IZE FFE were characterized according to molecular weight by Blue Native PAGE and were proteins stained with coomassie.

Down-regulation of ZmEXPB6 (Zea mays ß-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. Wall-loosening is a prerequisite for cell wall expansion, a process that is under the control of cell wall-located expansin proteins. In this study the abundance of those proteins was analyzed against salt stress using gel-based two-dimensional proteomics and two-dimensional Western blotting. Results show that ZmEXPB6 (Z. mays ß-expansin 6) protein is lacking in growth-inhibited leaves of salt-stressed maize. Of note, the exogenous application of heterologously expressed and metal-chelate-affinity chromatography-purified ZmEXPB6 on growth-reduced leaves that lack native ZmEXPB6 under NaCl stress partially restored leaf growth. In vitro assays on frozen-thawed leaf sections revealed that recombinant ZmEXPB6 acts on the capacity of the walls to extend. Our results identify expansins as a factor that partially restores leaf growth of maize in saline environments.

Localisation and interactions of the Vipp1 protein in cyanobacteria.

The Vipp1 protein is essential in cyanobacteria and chloroplasts for the maintenance of photosynthetic function and thylakoid membrane architecture. To investigate its mode of action we generated strains of the cyanobacteria Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942 in which Vipp1 was tagged with green fluorescent protein at the C-terminus and expressed from the native chromosomal locus. There was little perturbation of function. Live-cell fluorescence imaging shows dramatic relocalisation of Vipp1 under high light. Under low light, Vipp1 is predominantly dispersed in the cytoplasm with occasional concentrations at the outer periphery of the thylakoid membranes. High light induces Vipp1 coalescence into localised puncta within minutes, with net relocation of Vipp1 to the vicinity of the cytoplasmic membrane and the thylakoid membranes. Pull-downs and mass spectrometry identify an extensive collection of proteins that are directly or indirectly associated with Vipp1 only after high-light exposure. These include not only photosynthetic and stress-related proteins but also RNA-processing, translation and protein assembly factors. This suggests that the Vipp1 puncta could be involved in protein assembly. One possibility is that Vipp1 is involved in the formation of stress-induced localised protein assembly centres, enabling enhanced protein synthesis and delivery to membranes under stress conditions.

Isolation and characterisation of two cDNAs encoding transglutaminase from Atlantic cod (Gadus morhua).

Two cDNAs encoding transglutaminase (TG) were identified in a subtractive cDNA library prepared from the head kidney of poly I:C stimulated Atlantic cod (Gadus morhua). Full-length TG-1 and TG-2 cDNA were cloned from the head kidney by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The deduced amino acid (aa) sequence for TG-1 was 695 aa with an estimated molecular mass of 78.3 kDa, while TG-2 was a 698 aa protein with an estimated molecular mass of 78.8 kDa. The two proteins were named TG-1 and TG-2 and both possess transglutaminase/protease-like homologous domains (TGc) and full conservation of amino acids cysteine, histidine, and aspartate residues that form the catalytic triad. Sequence analysis showed high similarity (93.1%) with Alaska pollock TG, and the TGs were grouped together with TGs from chum salmon, Japanese flounder, Nile tilapia, and red sea bream in addition to Alaska pollock in phylogenetic analysis. Interestingly, they showed different tissue distribution with highest constitutive expression in reproductive and immunological organs, indicating important roles in these organs. Furthermore, the up-regulation of TG-1 and TG-2 in head kidney after stimulating Atlantic cod with poly I:C suggested a role of TGs in immune response in Atlantic cod.

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). Here, non-covalently bound chlorophyll is used as a sensitive probe to characterize the assembly/biogenesis of the pigment-protein complexes essential for photosynthesis. The high fluorescence yield recorded from chlorophyll-binding protein complexes can also be used to establish the separation of native protein complexes as an electrophoretic standard.

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. To begin to address this we undertook a proteomic analysis of three accumulation and replication of chloroplasts mutants that show a spectrum of plastid division perturbations. We show that defects in the chloroplast division process results in changes in the abundance of proteins when compared to wild type, but that the profile of the native stromal and membrane complexes remains unchanged. Furthermore, by combining BN-PAGE with protein interaction assays we show that AtFtsZ2-1 and AtFtsZ2-2 assemble together with rpl12A and EF-Tu into a novel chloroplast membrane complex.

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. Plants interact specifically with light via protein-bound pigments. This can be used in combination with CyDye technology to extend the "classical" approach in plant research. As an example, chlorophyll can be excited for fluorescent scanning at the Cy5 excitation wavelength. This property can be used to identify pigment-binding plant complexes and complex subunits isolated from plastid membranes. In this protocol, we present a combination of the conventional CyDye labeling technique with 2D native/SDS PAGE and parallel scanning for CyDyes and fluorescence from endogenous bound chlorophyll for identification of pigment-binding complexes and complex subunits.

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. The complex regulatory function in both developmental states is discussed.

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. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and ß-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.