Harald Paulsen

Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed in Escherichia coli.

A gene for a light-harvesting chlorophyll (Chl) a/b-binding protein (LHCP) from pea (Pisum sativum L.) has been cloned in a bacterial expression vector. Bacteria (Escherichia coli) transformed with this construct produced up to 20% of their protein as pLHCP, a derivative of the authentic precursor protein coded for by the pea gene with three amino-terminal amino acids added and-or exchanged, or as a truncated LHCP carrying a short amino-terminal deletion into the mature protein sequence. Following the procedure of Plumley and Schmidt (1987, Proc. Natl. Acad. Sci. USA84, 146–150), all bacteria-produced LHCP derivatives can be reconstituted with acetone extracts from pea thylakoids or with isolated pigments to yield pigment-protein complexes that are stable during partially denaturing polyacrylamide-gel electrophoresis. The spectroscopic properties of these complexes closely resemble those of the light-harvesting complex associated with photosystem II (LHCII) isolated from pea thylakoids. The pigment requirement for the reconstitution is highly specific for the pigments found in native LHCII: Chl a and b as well as at least two out of three xanthophylls are necessary. Varying the Chl a:Chl b ratios in the reconstitution mixtures changes the yields of complex formed but not the Chl a:Chl b ratio in the complex. We conclude that LHCP-pigment assembly in vitro is highly specific and that the complexes formed are structurally similar to LHCII. The N-terminal region of the protein can be varied without affecting complex formation and therefore does not seem to be involved in pigment binding.

Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex.

The major light‐harvesting complex (LHCII) of photosystem II, the most abundant chlorophyll‐containing complex in higher plants, is organized in trimers. In this paper we show that the trimerization of LHCII occurs spontaneously and is dependent on the presence of lipids. LHCII monomers were reconstituted from the purified apoprotein (LHCP), overexpressed in Escherichia coli, and pigments, purified from chloroplast membranes. These synthetic LHCII monomers trimerize in vitro in the presence of a lipid fraction isolated from pea thylakoids. The reconstituted LHCII trimers are very similar to native LHCII trimers in that they are stable in the presence of mild detergents and can be isolated by partially denaturing gel electrophoresis or by centrifugation in sucrose density gradients. Moreover, both native and reconstituted LHCII trimers exhibit signals in circular dichroism in the visible range that are not seen in native or reconstituted LHCII monomers, indicating that trimer formation either establishes additional pigment‐pigment interactions or alters pre‐existing interactions. Reconstituted LHCII trimers readily form two‐dimensional crystals that appear to be identical to crystals of the native complex.

Translocation of proteins into isolated chloroplasts requires cytosolic factors to obtain import competence

The precursor form of the major‐harvesting chlorophyll a/b‐binding protein (pLHCP) of chloroplast thylakoids was overproduced in E. coli cells and used to study the influence of soluble factors on post‐translational protein import into isolated pea chloroplasts. pLHCP solubilised in 8 M urea was not import‐competent. However, if pLHCP was dialysed in the presence of soluble proteins (leaf extract) after urea treatment, import competence was gained. Dialysis of pLHCP in the presence of leaf extract alters its protease sensitivity. Stremai proteins, ovalbumin, trypsin inhibitor or chloroplast lipids could not produce import competence of pLHCP. Two components from leaf extract seem to be necessary, one of which can be mimicked by purified hsc 70, the other one requiring ATP. We conclude that soluble proteins from outside the stromal compartment are necessary for post‐translational import of proteins into chloroplasts.

Topological arrangement of two transfer RNAs on the ribosome: Fluorescence energy transfer measurements between A and P site-bound tRNAPhe

The relative arrangement of two tRNAPhe molecules bound to the A and P sites of poly(U)-programmed Escherichia coli ribosomes was determined from the spatial separation of various parts of the two molecules. Intermolecular distances were calculated from the fluorescence energy transfer between fluorophores in the anticodon and D loops of yeast tRNAPhe. The energy donors were the natural fluorescent base wybutine in the anticodon loop or proflavine in both anticodon (position 37) and D loops (positions 16 and 17). The corresponding energy acceptors were proflavine or ethidium, respectively, at the same positions. Four distances were measured: anticodon loop-anticodon loop, 24(+/- 4) A; anticodon loop (A site)-D loop (P site), 46(+/- 12) A: anticodon loop (P site)-D loop (A site), 38(+/- 10) A: D loop-D loop, 35(+/- 9) A. Assuming that both tRNAs adopt the conformation present in the crystal and that the CCA ends are close to each other, the results are consistent with the two anticodons being bound to contiguous codons and suggest an asymmetric arrangement in which the planes of the two L-shaped molecules enclose an angle of 60 degrees +/- 30 degrees.

Pre-steady-state kinetics of ribosomal translocation☆

The two partial reactions of elongation factor G dependent translocation, the release of deacylated tRNA from the P site and the displacement of peptidyl tRNA from the A to the P site, have been studied with the stopped-flow technique. The experiments were performed with poly(U)-programmed ribosomes from Escherichia coli carrying deacylated tRNAPhe in the P site and N-AcPhe-tRNAPhe in the A site in the presence of GTP. The kinetics of the reaction were followed by monitoring either the intensity or the polarization of the fluorescence of both wybutine and proflavine located in the anticodon loop or of proflavine located in the D loop of yeast tRNAPhe or N-AcPhe-tRNAPhe. Both displacement and release fluorescence changes could be described by three exponentials, exhibiting apparent first-order rate-constants (20 degrees C) of 2 to 5 s-1 (15 s-1, 35 degrees C), 0.1 to 0.3 s-1, and 0.01 to 0.02 s-1, measured with a saturating concentration of elongation factor G (1 microM). The activation energy for the fast process of both reactions was found to be 70 kJ/mol (17 kcal/mol), while the intermediate process exhibits an activation energy of 30 kJ/mol (7 kcal/mol). The fast step is assigned to the displacement of the N-AcPhe-tRNAPhe from the A to the P site, and to the release of the tRNAPhe from the P site. The reactions take place simultaneously to form an intermediate post-translocation complex. The latter, in the intermediate step, rearranges to form a post-translocation complex carrying the deacylated tRNAPhe in an exit site and N-AcPhe-tRNAPhe in the P site, both in their equilibrium states. In parallel, or subsequently, the deacylated tRNAPhe spontaneously dissociates from the ribosome, thus completing the translocation process. The slow process has not been assigned.

Low-temperature spectroscopy of monomeric and trimeric forms of reconstituted light-harvesting chlorophyll a/b complex.

Low-temperature (polarized) light spectroscopy was used to study reconstituted light-harvesting complex (LHCII) of Photosystem II, both in the monomeric and trimeric form. Monomeric LHCII was reconstituted from the apoprotein overexpressed in Escherichia coli and pigments were extracted from chloroplast membranes and subsequently separated from unbound pigments on an anion-exchange column. These monomers trimerize in the presence of a lipid fraction isolated from thylakoids or of pure phosphatidylglycerol. The spectroscopic properties are compared to those of monomeric and trimeric forms of native LHCII and many similarities exist. However, these reconstituted complexes seem to contain slightly fewer chlorophylls, whereas one pigment that is a chlorophyll a in native LHCII is replaced by a chlorophyll b in reconstituted LHCII.

Light-Harvesting Chlorophyll a/b-Binding Protein Inserted into Isolated Thylakoids Binds Pigments and Is Assembled into Trimeric Light-Harvesting Complex

The light-harvesting chlorophyll a/b-binding protein (LHCP) is largely protected against protease (except for about 1 kD on the N terminus) in the thylakoid membrane; this protease resistance is often used to assay successful insertion of LHCP into isolated thylakoids in vitro. In this paper we show that this protease resistance is exhibited by trimeric light-harvesting complex of photosystem II (LHCII) but not by monomeric LHCII in which about 5 kD on the N terminus of LHCP are cleaved off by protease. When a mutant version of LHCP that is unable to trimerize in an in vitro reconstitution assay is inserted into isolated thylakoids, it gives rise to only the shorter protease digestion product indicative of monomeric LHCII. We conclude that more of the N-terminal domain of LHCP is shielded in trimeric than in monomeric LHCII and that this difference in protease sensitivity can be used to distinguish between LHCP assembled in LHCII monomers or trimers. The data presented prove that upon insertion of LHCP into isolated thylakoids at least part of the protein spontaneously binds pigments to form LHCII, which then is assembled in trimers. The dependence of the protease sensitivity of thylakoid-inserted LHCP on the oligomerization state of the newly formed LHCII justifies caution when using a protease assay to verify successful insertion of LHCP into the membrane.

PIGMENT COMPLEXES OF LIGHT‐HARVESTING CHLOROPHYLL a/b BINDING PROTEIN ARE STABILIZED BY A SEGMENT IN THE CARBOXYTERMINAL HYDROPHILIC DOMAIN OF THE PROTEIN

In order to identify segments of light‐harvesting chlorophyll a/6‐binding protein (LHCP) that are important for pigment binding, we have tested various LHCP mutants regarding their ability to form stable pigment‐protein complexes in an in vitro reconstitution assay. Deletion of 10 C‐terminal amino acids in the LHCP precursor, pLHCP, did not significantly affect pigment binding, whereas deletion of one additional amino acid, a tryptophan, completely abolished the formation of stable pigment‐protein complexes. This tryptophan, however, can be exchanged with other amino acids in full‐length pLHCP without noticeably altering the stability or spectroscopic properties of pigment complexes made with these mutants. Thus, the tryptophan residue is not likely to be involved in a highly specific interaction stabilizing the complex. A double mutant of LHCP lacking 66 N‐terminal and 6 C‐terminal amino acids still forms pigmented complexes that are virtually identical to those formed with the full‐length protein concerning their pigment composition and spectroscopic properties. We conclude that about 30% of the polypeptide chain in LHCP is not involved in pigment binding.

Carotenoids and the Assembly of Light-harvesting Complexes

Carotenoids are constitutive components of all light-harvesting complexes in plants and many such complexes in bacteria. In the crystal structures of several light-harvesting complexes, carotenoids are seen to span the lipid bilayer and connect components of the complex on both membrane surfaces and/or to mediate the interaction of transmembrane protein helices. This important stabilizing function suggests that these pigments are also actively involved in the assembly of light-harvesting complexes. Verification of this notion appears too ambitious a goal at present, as the question of how the pigment-protein complexes of the photosynthetic apparatus are assembled is still open. However, information is emerging about which light-harvesting complexes depend on the presence of carotenoids during their assembly, and which carotenoids are specifically required. This information comes from experiments in which allor some carotenoids are missing during biogenesis of the photosynthetic apparatus, due either to inhibitors of carotenoid biosynthesis or mutations in carotenoid biosynthesis pathways. Further information comes from reconstitution experiments in vitro in which light-harvesting complexes are assembled from their apoproteins and a pigment mixture containing a restricted or heterologous selection of carotenoids.

Lack of light regulation of NADPH : protochlorophyllide oxido-reductase mRNA in cress seedlings (Lepidium sativum L.)

Abstract A cDNA clone for NADPH: protochlorophyllide oxidoreductase from barley was subcloned for production of antisense-mRNA. This enabled heterologous hybridi zation with RNA from cress seedlings (Lepidium sativum L.). The m RNA level for NADPH: protochlorophyllide oxidoreductase did not decrease in cress seedlings during irradiation with continuous far-red or white light up to 12 h. The amount of NADPH: protochlorophyllide oxi doreductase protein, identified by Western blot de creased 5-fold after continuous irradiation with white light for 12 h. Species differences for light regulation of RNA are discussed.