The presence of multidomain linkers determines the bundle-shape structure of the phycobilisome of the cyanobacterium Gloeobacter violaceus PCC 7421

The complete genome sequence of Gloeobacter violaceus [Nakamura et al. (2003a, b) DNA Res 10:37–45, 181–201] allows us to understand better the structure of the phycobilisomes (PBS) of this cyanobacterium. Genomic analysis revealed peculiarities in these PBS: the presence of genes for two multidomain linker proteins, a core membrane linker with four repetitive sequences (REP domains), the absence of rod core linkers, two sets of phycocyanin (PC) α and β subunits, two copies of a rod PC associated linker (CpcC), and two rod cap associated linkers (CpcD). Also, there is one ferredoxin–NADP⁺ oxidoreductase with only two domains. The PBS proteins were investigated by gel electrophoresis, amino acid sequencing and peptide mass fingerprinting (PMF). The two unique multidomain linkers contain three REP domains with high similarity and these were found to be in tandem and were separated by dissimilar Arms. One of these, with a mass of 81 kDa, is found in heavy PBS fragments rich in PC. We propose that it links six PC hexamers in two parallel rows in the rods. The other unique linker has a mass of 91 kDa and is easily released from the heavy fragments of PBS. We propose that this links the rods to the core. The presence of these multidomain linkers could explain the bundle shaped rods of the PBS. The presence of 4 REP domains in the core membrane linker protein (129 kDa) was established by PMF. This core linker may hold together 16 AP trimers of the pentacylindrical core, or alternatively, a tetracylindrical core of the PBS of G. violaceus.     

New linker proteins in phycobilisomes isolated from the cyanobacterium Gloeobacter violaceus PCC 7421

Two new linker proteins were identified by peptide mass fingerprinting in phycobilisomes isolated from the cyanobacterium Gloeobacter violaceus PCC 7421. The proteins were products of glr1262 and glr2806. Three tandem phycocyanin linker motifs similar to CpcC were present in each. The glr1262 product most probably functions as a rod linker connecting phycoerythrin and phycocyanin, while the glr2806 product may function as a rod-core linker. We have designated these two proteins CpeG and CpcJ, respectively. The morphology of phycobilisomes in G. violaceus has been reported to be a bundle-like shape with six rods, consistent with the proposed functions of these linkers.     

High-resolution crystal structures of trimeric and rod phycocyanin

The phycobilisome light-harvesting antenna in cyanobacteria and red algae is assembled from two substructures: a central core composed of allophycocyanin surrounded by rods that always contain phycocyanin (PC). Unpigmented proteins called linkers are also found within the rods and core. We present here two new structures of PC from the thermophilic cyanobacterium Thermosynechococcus vulcanus. We have determined the structure of trimeric PC to 1.35 Å, the highest resolution reported to date for this protein. We also present a structure of PC isolated in its intact and functional rod form at 1.5 Å. Analysis of rod crystals showed that in addition to the α and β PC subunit, there were three linker proteins: the capping rod linker (L_R^8.7), the rod linker (L_R), and only one of three rod-core linkers (L_RC, CpcG4) with a stoichiometry of 12:12:1:1:1. This ratio indicates that the crystals contained rods composed of two hexamers. The crystallographic parameters of the rod crystals are nearly identical with that of the trimeric form, indicating that the linkers do not affect crystal packing and are completely embedded within the rod cavities. Absorption and fluorescence emission spectra were red-shifted, as expected for assembled rods, and this could be shown for the rod in solution as well as in crystal using confocal fluorescence microscopy. The crystal packing imparts superimposition of the three rod linkers, canceling out their electron density. However, analysis of B-factors and the conformations of residues facing the rod channel indicate the presence of linkers. Based on the experimental evidence presented here and a homology-based model of the L_R protein, we suggest that the linkers do not in fact link between rod hexamers but stabilize the hexameric assembly and modify rod energy absorption and transfer capabilities.     

Crystal structure of the N-terminal domain of linker L(R) and the assembly of cyanobacterial phycobilisome rods

Phycobilisomes are light‐harvesting supramolecular complexes in cyanobacteria and red algae. Linkers play a pivotal role in the assembly and energy transfer modulation of phycobilisomes. However, how linkers function remains unclear due to the lack of structural and biochemical studies of linkers, especially the N‐terminal domain of L_R (pfam00427). Here, we report the crystal structure of the pfam00427 domain of the linker L_R³⁰ from Synechocystis sp. PCC 6803 at 1.9 Å. The pfam00427 presents as a previously uncharacterized point symmetric six α‐helix bundle. To elucidate the binding style of pfam00427 in the C‐phycocyanin (C‐PC) (αβ)₆ hexamer, we fixed pfam00427 computationally into the C‐PC (αβ)₆ inner cavity using the program AutoDock. Combined with a conserved ‘C‐PC binding patch’ on pfam00427 identified, we arrived at a model for the pfam00427–C‐PC (αβ)₆ complex. This model was further optimized and evaluated as a reasonable result by a molecular dynamics simulation. In the resulting model, the pfam00427 domain is stably positioned in the central hole of the C‐PC trimer. Moreover, the L_RT (pfam01383) was docked into our pfam00427–C‐PC model to generate a complete phycobilisome rod in which the linkers join individual biliprotein hexamers.     

The phycocyanin-associated rod linker proteins of the phycobilisome of Gloeobacter violaceus PCC 7421 contain unusually located rod-capping domains

Gloeobacter violaceus PCC 7421 is a unique cyanobacterium that has no thylakoids and whose genome has been sequenced [Y. Nakamura, T. Kaneko, S. Sato, M. Mimuro, H. Miyashita, T. Tsuchiya, S. Sasamoto, A. Watanabe, K. Kawashima, Y. Kishida, C. Kiyokawa, M. Kohara, M. Matsumoto, A. Matsuno, N. Nakazaki, S. Shimpo, C. Takeuchi, M. Yamada, S. Tabata, Complete Genome Structure of Gloeobacter violaceus PCC 7421, a cyanobacterium that lacks thylakoids. DNA Research 10 (2003) 137-145]. Phycobilisomes of G. violaceus were isolated and analyzed by SDS-PAGE followed by N-terminal sequencing. Three rod-linker subunits (CpeC, CpeD and CpeE) were identified as predicted from the genome sequence. The cpcC1 and cpcC2 genes at order locus named (OLN) glr0950 and gll 3219 encoding phycocyanin-associated linker proteins from G. violaceus are 56 and 55 amino acids longer at the N-terminus than the open reading frame proposed in the genome. The two amino acid extensions showed a 66% identity to one another. Also, the N-terminal extensions of these sequences were similar to domains in both the rod-capping-linker protein CpcD2 and to the C-terminus domain of the phycoerythrin-associated linker protein CpeC. These domains are not only unusual in their N-terminal location, but are unusual in that they are more closely related in sequence similarity to the C-terminus domain of the phycoerythrin-associated linker, CpeC of G. violaceus, than to the C-terminus domain of phycocyanin-associated linker CpcC in other cyanobacteria. These linker proteins with unique special domains are indicators of the unusual structure of the phycobilisomes of G. violaceus.     

Single molecule study of the intrinsically disordered FG-repeat nucleoporin 153

Nucleoporins (Nups), which are intrinsically disordered, form a selectivity filter inside the nuclear pore complex, taking a central role in the vital nucleocytoplasmic transport mechanism. These Nups display a complex and nonrandom amino-acid architecture of phenylalanine glycine (FG)-repeat clusters and intra-FG linkers. How such heterogeneous sequence composition relates to function and could give rise to a transport mechanism is still unclear. Here we describe a combined chemical biology and single-molecule fluorescence approach to study the large human Nup153 FG-domain. In order to obtain insights into the properties of this domain beyond the average behavior, we probed the end-to-end distance (R_E) of several ∼50-residues long FG-repeat clusters in the context of the whole protein domain. Despite the sequence heterogeneity of these FG-clusters, we detected a reoccurring and consistent compaction from a relaxed coil behavior under denaturing conditions (R_E/R_E,RC = 0.99 ± 0.15 with R_E,RC corresponding to ideal relaxed coil behavior) to a collapsed state under native conditions (R_E/R_E,RC = 0.79 ± 0.09). We then analyzed the properties of this protein on the supramolecular level, and determined that this human FG-domain was in fact able to form a hydrogel with physiological permeability barrier properties.     

Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation

Summary The phycobilisome rod linker genes in the two closely related cyanobacteria Synechococcus sp. PCC 6301 and Synechococcus sp. PCC 7942 were studied. Southern blot analysis showed that the genetic organization of the phycobilisome rod operon is very similar in the two strains. The phycocyanin gene pair is duplicated and separated by a region of about 2.5 kb. The intervening region between the duplicated phycocyanin gene pair was cloned from Synechococcus sp. PCC 6301 and sequenced. Analysis of this DNA sequence revealed the presence of three open reading frames corresponding to 273, 289 and 81 amino acids, respectively. Insertion of a kanamycin resistance cassette into these open reading frames indicated that they corresponded to the genes encoding the 30, 33 and 9 kDa rod linkers, respectively, as judged by the loss of specific linkers from the phycobilisomes of the insertional mutants. Amino acid compositions of the 30 and 33 kDa linkers derived from the DNA sequence were found to deviate from those of purified 33 and 30 kDa linkers in the amounts of glutamic acid/glutamine residues. On the basis of similarity of the amino acid sequence of the rod linkers between Synechococcus sp. PCC 6301 and Calothrix sp. PCC 7601 we name the genes encoding the 30, 33 and 9 kDa linkers cpcH, cpcI and cpcD, respectively. The three linker genes were found to be co-transcribed on an mRNA of 3700 nucleotides. However, we also detected a smaller species of mRNA, of 3400 nucleotides, which would encode only the cpcH and cpcI genes. The 30 kDa linker was still found in phycobilisome rods lacking the 33 kDa linker and the 9 kDa linker was detected in mutants lacking the 33 or the 30 kDa linkers. Free phycocyanin was found in the mutants lacking the 33 or the 30 kDa linkers, whereas no free phycocyanin could be found in the mutant lacking the 9 kDa linker.Abbreviations PCC Pasteur Culture Collection - UTEX University of Texas Culture Collection The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank Nucleotide Sequence Databases under the accession number M94218     

DNA Sequence-Specific Ligands: XI.* The Synthesis and Binding to DNA of bis-Netropsins with the C-Ends of Their Netropsin Fragments Tethered by Tetra- or Pentamethylene Linkers

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A · T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5"-A ₄ T ₄-3" sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5"-T ₄ A ₄-3" sequences.     

The phycobilisome core-membrane linkers from Synechocystis sp. PCC 6803 and red-algae assemble in the same topology

The phycobilisomes (PBSs) of cyanobacteria and red-algae are unique megadaltons light-harvesting protein-pigment complexes that utilize bilin derivatives for light absorption and energy transfer. Recently, the high-resolution molecular structures of red-algal PBSs revealed how the multi-domain core-membrane linker (L_CM) specifically organizes the allophycocyanin subunits in the PBS’s core. But, the topology of L_CM in these structures was different than that suggested for cyanobacterial PBSs based on lower-resolution structures. Particularly, the model for cyanobacteria assumed that the Arm2 domain of L_CM connects the two basal allophycocyanin cylinders, whereas the red-algal PBS structures revealed that Arm2 is partly buried in the core of one basal cylinder and connects it to the top cylinder. Here, we show by biochemical analysis of mutations in the apcE gene that encodes L_CM, that the cyanobacterial and red-algal L_CM topologies are actually the same. We found that removing the top cylinder linker domain in L_CM splits the PBS core longitudinally into two separate basal cylinders. Deleting either all or part of the helix-loop-helix domain at the N-terminal end of Arm2, disassembled the basal cylinders and resulted in degradation of the part containing the terminal emitter, ApcD. Deleting the following 30 amino-acids loop severely affected the assembly of the basal cylinders, but further deletion of the amino-acids at the C-terminal half of Arm2 had only minor effects on this assembly. Altogether, the biochemical data are consistent with the red-algal L_CM topology, suggesting that the PBS cores in cyanobacteria and red-algae assemble in the same way.     

Seasonal and spatial variation of woody tissue respiration in a <Emphasis Type="Italic">Pinus cembra</Emphasis> tree at the alpine timberline in the central Austrian Alps

Total stem, branch, twig, and coarse root respiration (R_t) of an adult Pinus cembra tree at the alpine timberline was measured continuously at ten positions from 7 October 2001 to 21 January 2003 with an automated multiplexing gas exchange system. There was a significant spatial variability in woody tissue respiration when expressed per unit surface area or per unit sapwood volume. Surface area related maintenance (R_m) respiration at 0°C ranged between 0.109 and 0.643 mol m^–2 s^–1 and there was no clear trend with respect to tissue type and diameter. Sapwood volume based R_m at 0°C by contrast, varied between 2.5 mol m^–3 s^–1 in the stem and 193.2 mol m^–3 s^–1 in thin twigs in the upper crown. Estimated Q₁₀ values ranged from 1.7 to 3.1. These Q₁₀ values were used along with R_m at 0°C and annual woody tissue temperature records to predict annual total R_m. Annual total R_m accounted for 73±6% of annual R_t in 2002.     

Effect of interdomain dynamics on the structure determination of modular proteins by small-angle scattering

Multidomain proteins in which consecutive globular regions are connected by linkers are prevalent in nature (Levitt in Proc Natl Acad Sci USA 106:11079–11084, 2009). Some members of this family have largely resisted structural characterization as a result of challenges associated with their inherent flexibility. Small-angle scattering (SAS) is often the method of choice for their structural study. An extensive set of simulated data for both flexible and rigid multidomain systems was analyzed and modeled using standard protocols. This study clearly shows that SAXS profiles obtained from highly flexible proteins can be wrongly interpreted as arising from a rigid structure. In this context, it would be important to identify features from the SAXS data or from the derived structural models that indicate interdomain motions to differentiate between these two scenarios. Features of SAXS data that identify flexible proteins are: (1) general attenuation of fine structure in the scattering profiles, which becomes more dramatic in Kratky representations, and (2) a reduced number of interdomain correlation peaks in p(r) functions that also present large D _max values and a smooth decrease to 0. When modeling this dynamically averaged SAXS data, the structures obtained present characteristic trends: (1) ab initio models display a decrease in resolution, and (2) rigid-body models present highly extended conformations with a lack of interdomain contacts. The ensemble optimization method represents an excellent strategy to identify interdomain motions unambiguously. This study provides information that should help researchers to select the best modeling strategy for the structural interpretation of SAS experiments of multidomain proteins.     

Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L. cv. Poinsett76) via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, 1962) medium supplemented with 1 mg L⁻¹ benzyladenine (BA), 20 mg L⁻¹ l-glutamine and 2 mg L⁻¹ phosphinothricin (PPT) or 100 mg L⁻¹ kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2 mg L⁻¹) containing MS media with 0.5 mg L⁻¹ gibberellic acid (GA₃) and 0.6 mg L⁻¹ indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T₀ and the progenies. T₁ segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants.     

Synthesis and Chiral Recognition of Nickel(II) Macrocyclic Complex with (R)‐Naphthylethyleneamine Pendant Groups and its Self‐Assembled Framework

A novel nickel(II) hexaaza macrocyclic complex, [Ni(L^R,R)](ClO₄)₂ ( 1 ), containing chiral pendant groups was synthesized by an efficient one‐pot template condensation and characterized (L^R,R═1,8‐di((R)‐α‐methylnaphthyl)‐1,3,6,8,10,13‐hexaazacyclotetradecane). The crystal structure of compound 1 was determined by single‐crystal X‐ray analysis. The complex was found to have a square‐planar coordination environment for the nickel(II) ion. Open framework [Ni(L^R,R)]₃[C₆H₃(COO)₃]₂ ( 2 ) was constructed from the self‐assembly of compound 1 with deprotonated 1,3,5‐benzenetricarboxylic acid, BTC^3?. Chiral discrimination of rac‐1,1′‐bi‐2‐naphthol and rac‐2,2,2‐trifluoro‐1‐(9‐anthryl)ethanol was performed to determine the chiral recognition ability of the chiral complex ( 1 ) and its self‐assembled framework ( 2 ). Binaphthol showed a good chiral discrimination on the framework ( 2 ). The optimum experimental conditions for the chiral discrimination were examined by changing the weight ratio between the macrocyclic complex 1 or self‐assembled framework 2 and racemates. The detailed synthetic procedures, spectroscopic data including single‐crystal X‐ray analysis, and the results of the chiral recognition for the compounds are described. Chirality, 25:54‐58, 2013 © 2012 Wiley Periodicals, Inc.     

SYNTHESIS AND BINDING OF PHYCOERYTHRIN AND ITS ASSOCIATED LINKERS TO THE PHYCOBILISOME IN RHODELLA VIOLACEA (RHODOPHYTA): COMPARED EFFECTS OF HIGH LIGHT AND TRANSLATION INHIBITORS1

We studied the synthesis and binding of phycoerythrin and its associated linkers to the phycobilisome (PBS) in Rhodella violacea (Kornmann) Wehrmeyer and compared the effects of high light and translation inhibitors on these processes. Rhodella violacea has a simple hemidiscoidal PBS structure with a well-known composition. The number of PBSs per cell decreases when irradiance is increased, and at higher irradiances the rods are shortened with a specific loss of the terminal hexamer of phycoerythrin (PE) and its associated linker. To test whether or not the observed variations were due to a coordination between the expression of the chloroplast-encoded PE and the nuclear-encoded linkers, we inhibited the expression of the chloroplast genes by the translation inhibitor chloramphenicol. In the few PBSs synthesized, the linker associated to the terminal PE hexamer was missing while that associated with the intermediate PE hexamer was still present. The inhibition by cycloheximide of the translation of the nuclear-encoded linkers did not influence the synthesis of the chloroplast-encoded phycobiliproteins. The absence of linkers prevented the formation of PE hexamers and their binding to the PBSs. We therefore propose the existence of two levels of regulation for PE and associated linkers: the intermediate PE hexamer and associated linker are always present even though their amount is reduced when irradiance is increased. In contrast, the terminal hexamer of PE and its associated linker are no longer present under high light. Their absence can be due to a feedback control between the level of PE and the synthesis of the linker: when the level of PE is lowered below a given value by the action of light on the chloroplast, a signal coming from the chloroplast reaches the nucleus and the synthesis of the linker is repressed. There is no sign of nuclear regulation of the synthesis of PE, but the nuclear-encoded linkers have a structural role in the formation of PE hexamers.     

Structure and mutation of a gene encoding a Mr 33000 phycocyanin-associated linker polypeptide

The gene encoding a phycocyanin-associated linker polypeptide of M_r 33000 from the cyanobacterium Synechococcus sp. PCC 7002 was found to be located adjacent and 3 to the genes encoding the and subunits of phycocyanin. The identity of this gene, designated cpcC, was proven by matching the amino-terminal sequence of the authentic polypeptide with that predicted by the nucleotide sequence. A cpcC mutant strain of this cyanobacterium was constructed. The effect of the mutation was to prevent assembly of half the total phycocyanin into phycobilisomes. By electron microscopy, phycobilisomes from this mutant were shown to contain rod substructures composed of a single disc of hexameric phycocyanin, as opposed to two discs in the wild type. It was concluded that the M_r 33000 linker polypeptide is required for attachment of the core-distal phycocyanin hexamer to the core-proximal one. Using absorption spectra of the wild type, CpcC^–, and phycocyanin-less phycobilisomes, the in situ absorbances expected for specific phycocyanin-linker complexes were calculated. These data confirm earlier findings on isolated complexes regarding the influence of linkers on the spectroscopic properties of phycocyanin.Abbreviations PC phycocyanin - PEC phycoerythrocyanin - AP allophycocyanin - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Linker polypeptides are abbreviated according to Glazer (1985). L _infX ^supY refers to a linker having a mass Y, located at a position X in the phycobilisome, where X can be R (rod), RC (rod or core), C (core) or CM (core to membrane). When necessary, the abbreviation for a linker is appended with that of its associated phycobiliprotein. Thus, L _infR ^sup34.5PEC is a rod linker of M_r 34 500 that is associated with phycoerythrocyanin     

Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 131-deoxo-131-hydroxy-pheophytin a and comparison of their 6 K absorption spectra

Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 13¹-deoxo-13¹-hydroxy-pheophytin a (13¹-OH-Pheo). The absorption bands of 13¹-OH-Pheo are blue-shifted and well separated from those of Pheo. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC_1×) or twice (RC_2×). HPLC analysis and Pheo Q_X absorption at 543 nm show that in RC_1× about 50% of Pheo is replaced and in RC_2× about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo⁻ is unmodified in RC_1× and decreased only in RC_2×, suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption changes in the Q_Y region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC_1× or RC_2×, showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of photosynthetic activity of <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> and oil-seed rape

Orychophragmus violaceus, Brassica campestris cv. Chuanyou No.8, and Brassica juncea cv. Luzhousileng diurnal changes of net photosynthetic rate (P _N) and activities of carbonic anhydrase (CA) of leaves were studied. One uni-modal curve occurred at the diurnal changes of P _N in O. violaceus, but bimodal curves were found in B. campestris and B. juncea. Thus photosynthetic midday depression was not found in O. violaceus but in both Brassica species. Midday depression of P _N in O. violaceus was not related to high temperature or low humidity at midday but to the activity of CA.