Single molecule spectroscopy on the light-harvesting complex II of higher plants.

Spectroscopic and polarization properties of single light-harvesting complexes of higher plants (LHC-II) were studied at both room temperature and T < 5 K. Monomeric complexes emit roughly linearly polarized fluorescence light thus indicating the existence of only one emitting state. Most probably this observation is explained by efficient triplet quenching restricted to one chlorophyll a (Chl a) molecule or by rather irreversible energy transfer within the pool of Chl a molecules. LHC-II complexes in the trimeric (native) arrangement bleach in a number of steps, suggesting localization of excitations within the monomeric subunits. Interpretation of the fluorescence polarization properties of trimers requires the assumption of transition dipole moments tilted out of the symmetry plane of the complex. Low-temperature fluorescence emission of trimers is characterized by several narrow spectral lines. Even at lowest excitation intensities, we observed considerable spectral diffusion most probably due to low temperature protein dynamics. These results also indicate weak interaction between Chls belonging to different monomeric subunits within the trimer thus leading to a localization of excitations within the monomer. The experimental results demonstrate the feasibility of polarization sensitive studies on single LHC-II complexes and suggest an application for determination of the Chl transition-dipole moment orientations, a key issue in understanding the structure-function relationships.     

The three-dimensional structure of frozen-hydrated Nudaurelia capensis β Virus, a T = 4 insect Virus

The three-dimensional structure of Nudaurelia capensis beta virus (N beta V) was reconstructed to 3.2-nm resolution from images of frozen-hydrated virions. The distinctly icosahedral capsid (approximately 40-nm diameter) contains 240 copies of a single 61-kDa protein subunit arranged with T = 4 lattice symmetry. The outer surface of unstained virions compares remarkably well with that previously observed in negatively stained specimens. Inspection of the density map, volume estimates, and model building experiments indicate that each subunit consists of two distinct domains. The large domain (approximately 40 kDa) has a cylindrical shape, approximately 4-nm diameter by approximately 4-nm high, and associates with two large domains of neighboring subunits to form a Y-shaped trimeric aggregate in the outer capsid surface. Four trimers make up each of the 20 planar faces of the capsid. Small domains (approximately 21 kDa) presumably associate at lower radii (approximately 13-16.5 nm) to form a contiguous, non-spherical shell. A T = 4 model, constructed from 80 trimers of the common beta-barrel core motif (approximately 20 kDa) found in many of the smaller T = 3 and pseudo T = 3 viruses, fits the dimensions and features seen in the N beta V reconstruction, suggesting that the contiguous shell of N beta V may be formed by intersubunit contacts between small domains having that motif. The small (approximately 1800 kDa), ssRNA genome is loosely packed inside the capsid with a low average density.     

Three-dimensional reconstruction of maltoporin from electron microscopy and image processing.

Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis. Two different trimer packing forms were observed. One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm). In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing. At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane. The pore defined by maltoporin has a similar structure to that outlined by the matrix protein. From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit. A tentative outline of the maltoporin promoter is given. Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.     

Structural Insight into concerted inhibition of alpha 2 beta 2-type aspartate kinase from Corynebacterium glutamicum

Aspartate kinase (AK) catalyzes the first step of the biosynthesis of the aspartic acid family amino acids, and is regulated via feedback inhibition by end-products including Thr and Lys. To elucidate the mechanism of this inhibition, we determined the crystal structure of the regulatory subunit of AK from Corynebacterium glutamicum at 1.58 A resolution in the Thr-binding form, the first crystal structure of the regulatory subunit of alpha(2)beta(2)-type AK. The regulatory subunit contains two ACT domain motifs per monomer and is arranged as a dimer. Two non-equivalent ACT domains from different chains form an effector-binding unit that binds a single Thr molecule, and the resulting two effector-binding units of the dimer associate perpendicularly in a face-to-face manner. The regulatory subunit is a monomer in the absence of Thr but becomes a dimer by adding Thr. The dimerization is eliminated in mutant AKs with changes in the Thr-binding region, suggesting that the dimerization induced by Thr binding is a key step in the inhibitory mechanism of AK from C. glutamicum. A putative Lys-binding site and the inhibitory mechanism of CgAK are discussed.     

X-ray crystal structure of a galactose-specific C-type lectin possessing a novel decameric quaternary structure

Rattlesnake venom lectin (RSL) from the western diamondback rattlesnake (Crotalus atrox) is an oligomeric galactose-specific C-type lectin. The X-ray crystal structure of RSL, in complex with lactose and thiodigalactoside, at 2.2 and 2.3 A resolution, respectively, reveals a decameric protein composed of two 5-fold symmetric pentamers arranged in a staggered, back-to-back orientation. Each monomer corresponds to a single canonical C-type lectin carbohydrate recognition domain devoid of accessory domains and is disulfide-bonded to a monomer in the other pentamer. The structure is the first example of that of a carbohydrate complex of a vertebrate galactose-specific C-type lectin. The 10 carbohydrate-binding sites, located on the rim of the decamer, suggest a role for multivalent interactions and a mechanism for RSL's ability to promote receptor cross-linking and cell aggregation.     

Trigonal crystals of porin from Escherichia coli

Trigonal crystals of the integral membrane protein porin from Escherichia coli have been grown and characterized. They belong to space group P321 with unit cell constants a = b = LL8.4, c = 52.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystals grow as well-defined hexagonal prisms to a size of 0.25 mm in all dimensions, and diffract to 2.7 A. The molecular symmetry coincides with 3-fold crystallographic symmetry, giving two trimers per unit cell (1 monomer/asymmetric unit). This corresponds to VM = 2.9 A3/Da. Native X-ray data to 3.0 A resolution have been collected on a FAST area detector and a search for heavy atom derivatives is underway.     

The structure of the multidrug resistance protein 1 (MRP1/ABCC1). crystallization and single-particle analysis

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) polytopic membrane transporter of considerable clinical importance that confers multidrug resistance on tumor cells by reducing drug accumulation by active efflux. MRP1 is also an efficient transporter of conjugated organic anions. Like other ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD). However, unlike P-glycoprotein and most other ABC superfamily members, MRP1 contains a third MSD with five predicted transmembrane segments with an extracytosolic NH(2) terminus. Moreover, the two nucleotide-binding domains of MRP1 are considerably more divergent than those of P-glycoprotein. In the present study, the first structural details of MRP1 purified from drug-resistant lung cancer cells have been obtained by electron microscopy of negatively stained single particles and two-dimensional crystals formed after reconstitution of purified protein with lipids. The crystals display p2 symmetry with a single dimer of MRP1 in the unit cell. The overall dimensions of the MRP1 monomer are approximately 80 x 100 A. The MRP1 monomer shows some pseudo-2-fold symmetry in projection, and in some orientations of the detergent-solubilized particles, displays a stain filled depression (putative pore) appearing toward the center of the molecule, presumably to enable transport of substrates. These data represent the first structural information of this transporter to approximately 22-A resolution and provide direct structural evidence for a dimeric association of the transporter in a reconstituted lipid bilayer.     

Crystal structure of allophycocyanin from red algae Porphyra yezoensis at 2.2-A resolution.

The crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) at 2.2-A resolution has been determined by the molecular replacement method. The crystal belongs to space group R32 with cell parameters a = b = 105.3 A, c = 189.4 A, alpha = beta = 90 degrees, gamma = 120 degrees. After several cycles of refinement using program X-PLOR and model building based on the electron density map, the crystallographic R-factor converged to 19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 A. The r.m.s. deviations of bond length and angles are 0.015 A and 2.9 degrees, respectively. In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers. The chromophore-protein interactions and possible pathway of energy transfer were discussed. Phycocyanobilin 1alpha84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2beta of another monomer. In R-PE and C-PC, chromophore 1alpha84 only forms 1 hydrogen bond with 2beta77 residue in subunit 2beta. This result may support and explain great spectrum difference exists between APC trimer and monomer.     

Crystal structure of a bifunctional transformylase and cyclohydrolase enzyme in purine biosynthesis

ATIC, the product of the purH gene, is a 64 kDa bifunctional enzyme that possesses the final two activities in de novo purine biosynthesis, AICAR transformylase and IMP cyclohydrolase. The crystal structure of avian ATIC has been determined to 1.75 A resolution by the MAD method using a Se-methionine modified enzyme. ATIC forms an intertwined dimer with an extensive interface of approximately 5,000 A(2) per monomer. Each monomer is composed of two novel, separate functional domains. The N-terminal domain (up to residue 199) is responsible for the IMPCH activity, whereas the AICAR Tfase activity resides in the C-terminal domain (200-593). The active sites of the IMPCH and AICAR Tfase domains are approximately 50 A apart, with no structural evidence of a tunnel connecting the two active sites. The crystal structure of ATIC provides a framework to probe both catalytic mechanisms and to design specific inhibitors for use in cancer chemotherapy and inflammation.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700(+) and Y(D)( .-), respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIbeta) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIalpha) 300, PSI (PSIbeta) in stroma lamellae 214, PSII in grana core (PSIIalpha) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

Sub-domain structure of lipid-bound annexin-V resolved by electron image analysis.

Two-dimensional crystals of annexin-V bound to lipid layers containing dioleoylphosphatidylserine have been obtained in the presence of Ca2+. The crystals diffract to 20 A resolution and have the symmetry of the plane group p3 (unit cell dimensions: a = b = 94 A, gamma = 120 degrees). Electron image analysis revealed that the crystals are composed of trimers of annexin-V forming triskelion-like motifs. Each annexin-V molecule has a characteristic elongated shape, about 65 A by 20 A, when observed perpendicularly to the crystal plane. It is composed of two staggered domains of similar size, about 40 A by 20 A. Both domains are made of two sub-domains. The present data suggest that the four resolved sub-domains represent the folding units corresponding to the four 70 amino acid repeating segments characteristic of all annexins.     

The crystal structure of muscle phosphoglucomutase refined at 2.7-angstrom resolution.

A model of rabbit muscle phosphoglucomutase was refined at 2.7-A resolution by using two heavy atom derivatives for initial phasing and standard refinement procedures, including molecular replacement averaging about a 2-fold axis and dynamic simulation: final R-factor, 0.223 (no solvent modeling); RMS deviation from standard bond lengths and angles, 0.020 A and 3.6 degrees, respectively (all 8658 nonhydrogen atoms plus 36,953 reflections (F/sigma greater than or equal to 3) between 8- and 2.7-A resolutions); average of individually refined atomic B-factors, 40 A2 (all atoms) and 30 A2 (all atoms in domains I-III). An H-bonding scheme with 538 main chain H-bonds for the two monomers in the asymmetric unit and probable ligands for six uranyl ions in one heavy atom derivative is given. The monomer contains 42 strands/helices arranged into four alpha/beta-domains. Each of the first three domains contains an alpha 3 beta 4 alpha 1 motif, where the topology of beta 4 is 2,1,3,4:[arrows: see text] which is a topology not encountered in an extensive search among known protein structures. A spatial similarity is observed between corresponding residues in the three repetitions of this motif per monomer, but the minimal mutational distance between spatially corresponding residues is not statistically significant. The loop between the antiparallel strands in each of these domains is an important feature of the active site. In domain IV, beta-sheet topology is 2,1,3,4,5,6:[arrows:see text]. Noncovalent domain/domain interactions within the monomer are greatest between adjacent domains along the polypeptide chain, which are not substantially interdigitated and can be cleanly disengaged by altering the phi/psi torsional angles of three uniquely positioned residues in the model. The observed hierarchy of noncovalent interactions between structural units within the crystal, based on a semi-empirical paradigm, suggests that monomer-monomer contacts within the asymmetric unit are formed during growth of the lattice and provides a rationale for some of the diffraction characteristics of phosphoglucomutase crystals. An unusually deep crevice involving 58 residues is formed by the head-to-tail, twisted semicircular arrangement of the four domains of the monomer that places no atom more than 12 A from the water-accessible surface. The active site of the enzyme is extensively buried at the bottom of this crevice, at the approximate confluence of the four domains. Other features of the active site, including the surrounding helical dipoles, and the metal-ion binding pocket are described, together with structure/function comparisons with a number of other enzymes.     

Mutant trimers of light-harvesting complex II exhibit altered pigment content and spectroscopic features

Mutants of plant light-harvesting complex II (LHC-II) were produced by refolding the complex in vitro from bacterially expressed apoprotein and purified pigments by a method which yields native-like LHC-II in a single step. Amino acid residues known from the structure of the complex [Kühlbrandt, W., et al. (1994) Nature 367, 614-621] to bind chlorophyll (Chl) were replaced with nonbinding residues by site-directed mutagenesis. Recombinant monomeric and trimeric pigment-protein complexes were separated by density gradient centrifugation, and their pigment composition was determined. Six out of nine mutants formed trimers with Chl a:Chl b ratios and Chl contents which suggested they were lacking one Chl a or b per polypeptide. In this way, the identities of Chls a1, a2, a3, b5, and b6 were confirmed as Chl a or b, respectively, whereas Chl b3 in the structure was found to be a Chl a. Absorption and fluorescence emission spectra of the mutant lacking Chl a2 indicated a central role for this Chl in energy transfer to the reaction center.     

Purification of a protein phosphatase from chloroplast stroma capable of dephosphorylating the light-harvesting complex-II.

A protein phosphatase was purified from the stroma of Pea (Pisum sativum L.) chloroplasts that is capable of dephosphorylating synthetic phosphopeptides. Following chromatographic purification of greater than 400-fold, two-dimensional electrophoresis indicated that the stromal protein phosphatase is a 29-kD protein. A similar molecular size was determined for the protein-phosphatase activity using gel-permeation chromatography, indicating that the stromal protein phosphatase is probably a monomer. The purified enzyme was able to dephosphorylate synthetic phosphopeptides, which mimic the thylakoid light-harvesting complex II (LHC-II) N terminus, as well as LHC-II in thylakoid membranes, but did not dephosphorylate the major 64-kD phosphoprotein in the stroma. The stromal protein phosphatase did not discriminate between dephosphorylation of phosphothreonine and phosphoserine residues in synthetic peptide substrates, providing further evidence that this enzyme is distinct from the protein phosphatase localized in thylakoid membranes. The exact physiological role of the stromal protein phosphatase has yet to be determined, but it may function in the dephosphorylation of LHC-II.     

3D structure of the skeletal muscle dihydropyridine receptor

The dihydropyridine receptors (DHPR) are L-type voltage-gated calcium channels that regulate the flux of calcium ions across the cell membrane. Here we present the three-dimensional (3D) structure at approximately 27A resolution of purified skeletal muscle DHPR, as determined by electron microscopy and single particle analysis. Here both biochemical and 3D structural data indicate that DHPR is dimeric. DHPR dimers are composed of two arch-shaped monomers approximately 210A across and approximately 75A thick, that interact very tightly at each end of the arch. The roughly toroidal structure of the two monomers encloses a cylindrical space of approximately 80A diameter, which is then closed on each side by two dome-shaped protein densities reaching over from each monomer arch. The dome-shaped domains have a length of approximately 80-90A and a maximum height of approximately 45A. Small orifices punctuate their exterior surface. The 3D structure disclosed here may have important implications for the understanding of DHPR Ca(2+) channel function. We also propose a model for its in vivo interactions with the calcium release channel at the junctional sarcoplasmic recticulum.     

Molecular mechanism of in vitro oligomerization of Dps from Mycobacterium smegmatis: mutations of the residues identified by "interface cluster" analysis

The irreversible dodecamerization of native Dps trimers from Mycobacterium smegmatis, in vitro, is known to be directly associated with the bimodal function of this protein. Hence it is important to explore this pathway at the molecular level. Two types of trimers, Trimer A (tA) and Trimer B (tB), can be derived from the dodecamer due to the inherent 3-fold symmetry of the spherical crystal structure. These derived trimers were expressed as protein structure graphs (PSGs) using the computed interaction strength among the residues. Interface clusters which were identified from PSGs allowed us to convincingly predict E146 and F47 for further mutation studies. Various single and double mutants were constructed and characterized. We were finally able to generate a single mutant F47E impaired in dodecamerization and a double mutant E146AF47E as native monomer in solution. These two observed results suggest that the two trimers are important for dodecamerization and that the residues selected are important for the structural stability of the protein in vitro.     

Low-temperature energy transfer in LHC-II trimers from the Chl a/b light-harvesting antenna of photosystem II.

Temperature dependence in electronic energy transfer steps within light-harvesting antenna trimers from photosystem II was investigated by studying Chl a pump-probe anisotropy decays at several wavelengths from 675 to 682 nm. The anisotropy lifetime is markedly sensitive to temperature at the longest wavelengths (680-682 nm), increasing by factors of 5 to 6 as the trimers are cooled from room temperature to 13 K. The temperature dependence is muted at 677 and 675 nm. This behavior is modeled using simulations of temperature-broadened Chl a absorption and fluorescence spectra in spectral overlap calculations of Förster energy transfer rates. In this model, the 680 nm anisotropy decays are dominated by uphill energy transfers from 680 nm Chl a pigments at the red edge of the LHC-II spectrum; the 675 nm anisotropy decays reflect a statistical average of uphill and downhill energy transfers from 676-nm pigments. The measured temperature dependence is consistent with essentially uncorrelated inhomogeneous broadening of donor and acceptor Chl a pigments.     

Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A resolution

Colicin B (55 kDa) is a cytotoxic protein that recognizes the outer membrane transporter, FepA, as a receptor and, after gaining access to the cytoplasmic membranes of sensitive Escherichia coli cells, forms a pore that depletes the electrochemical potential of the membrane and ultimately results in cell death. To begin to understand the series of dynamic conformational changes that must occur as colicin B translocates from outer membrane to cytoplasmic membrane, we report here the crystal structure of colicin B at 2.5 A resolution. The crystal belongs to the space group C2221 with unit cell dimensions a = 132.162 A, b = 138.167 A, c = 106.16 A. The overall structure of colicin B is dumbbell shaped. Unlike colicin Ia, the only other TonB-dependent colicin crystallized to date, colicin B does not have clearly structurally delineated receptor-binding and translocation domains. Instead, the unique N-terminal lobe of the dumbbell contains both domains and consists of a large (290 residues), mostly beta-stranded structure with two short alpha-helices. This is followed by a single long ( approximately 74 A) helix that connects the N-terminal domain to the C-terminal pore-forming domain, which is composed of 10 alpha-helices arranged in a bundle-type structure, similar to the pore-forming domains of other colicins. The TonB box sequence at the N-terminus folds back to interact with the N-terminal lobe of the dumbbell and leaves the flanking sequences highly disordered. Comparison of sequences among many colicins has allowed the identification of a putative receptor-binding domain.     

Transcarboxylase 12S crystal structure: hexamer assembly and substrate binding to a multienzyme core

Transcarboxylase from Propionibacterium shermanii is a 1.2 MDa multienzyme complex that couples two carboxylation reactions, transferring CO(2)(-) from methylmalonyl-CoA to pyruvate, yielding propionyl-CoA and oxaloacetate. The 1.9 A resolution crystal structure of the central 12S hexameric core, which catalyzes the first carboxylation reaction, has been solved bound to its substrate methylmalonyl-CoA. Overall, the structure reveals two stacked trimers related by 2-fold symmetry, and a domain duplication in the monomer. In the active site, the labile carboxylate group of methylmalonyl-CoA is stabilized by interaction with the N-termini of two alpha-helices. The 12S domains are structurally similar to the crotonase/isomerase superfamily, although only domain 1 of each 12S monomer binds ligand. The 12S reaction is similar to that of human propionyl-CoA carboxylase, whose beta-subunit has 50% sequence identity with 12S. A homology model of the propionyl-CoA carboxylase beta-subunit, based on this 12S crystal structure, provides new insight into the propionyl-CoA carboxylase mechanism, its oligomeric structure and the molecular basis of mutations responsible for enzyme deficiency in propionic acidemia.     

Structure of the ATPase subunit CysA of the putative sulfate ATP-binding cassette (ABC) transporter from Alicyclobacillus acidocaldarius

CysA, the ATPase subunit of a putative sulfate ATP-binding cassette transport system of the gram-positive thermoacidophilic bacterium Alicyclobacillus acidocaldarius, was structurally characterized at a resolution of 2.0 Angstroms in the absence of nucleotides. In line with previous findings on ABC-ATPases the structures of the two monomers (called CysA-1 and CysA-2) in the asymmetric unit differ substantially in the arrangement of their individual (sub)domains. CysA-2 was found as a physiological dimer composed of two crystallographically related monomers that are arranged in an open state. Interestingly, while the regulatory domain of CysA-2 packs against its opposing domain that of CysA-1 undergoes a conformational change and, in the dimer, would interfere with the opposing monomer thereby preventing solute translocation. Whether this conformational state is used for regulatory purposes will be discussed.