Structural analysis of the reaction center light-harvesting complex I photosynthetic core complex of Rhodospirillum rubrum using atomic force microscopy

The bacterium Rhodospirillum rubrum contains a simple photosynthetic system, in which the reaction center (RC) receives energy from the light-harvesting (LH1) complex. We have used high-resolution atomic force microscopy (AFM) to image two-dimensional crystals of the RC-LH1 complex of R. rubrum. The AFM topographs show that the RC-LH1 complex is approximately 94 A in height, the RC-H subunit protrudes from the cytoplasmic face of the membrane by 40 A, and it sits 21 A above the highest point of the surrounding LH1 ring. In contrast, the RC on the periplasmic side is at a lower level than LH1, which protrudes from the membrane by 12 A. The RC-LH1 complex can adopt an irregular shape in regions of uneven packing forces in the crystal; this reflects a likely flexibility in the natural membrane, which might be functionally important by allowing the export of quinol formed as a result of RC photochemistry. Nanodissection of the RC by the AFM tip removes the RC-H subunit and reveals the underlying RC-L and -M subunits. LH1 complexes completely lacking the RC were also found, providing ideal conditions for imaging both rings of LH1 polypeptides for the first time by AFM. In addition, we demonstrate the ellipticity of the LH1 ring at the cytoplasmic and periplasmic sides of the membrane, in both the presence and absence of the RC. These AFM measurements have been reconciled with previous electron microscopy and NMR data to produce a model of the RC-LH1 complex.     

Projection structure of the photosynthetic reaction centre-antenna complex of Rhodospirillum rubrum at 8.5 A resolution

Two-dimensional crystals of the reaction-centre-light-harvesting complex I (RC-LH1) of the purple non- sulfur bacterium Rhodospirillum rubrum have been formed from detergent-solubilized and purified protein complexes. Unstained samples of this intrinsic membrane protein complex have been analysed by electron cryomicroscopy (cryo EM). Projection maps were calculated to 8.5 A from two different crystal forms, and show a single reaction centre surrounded by 16 LH1 subunits in a ring of approximately 115 A diameter. Within each LH1 subunit, densities for the alpha- and beta-polypeptide chains are clearly resolved. In one crystal form the LH1 forms a circular ring, and in the other form the ring is significantly ellipsoidal. In each case, the reaction centre adopts preferred orientations, suggesting specific interactions between the reaction centre and LH1 subunits rather than a continuum of possible orientations with the antenna ring. This experimentally determined structure shows no evidence of any other protein components in the closed LH1 ring. The demonstration of circular or elliptical forms of LH1 indicates that this complex is likely to be flexible in the bacterial membrane.     

Role of the C-terminal extrinsic region of the alpha polypeptide of the light-harvesting 2 complex of Rhodobacter sphaeroides: a domain swap study

The LH1 and LH2 complexes of Rhodobacter sphaeroides form ring structures of 16 and 9 protomers, respectively, comprising alpha and beta polypeptides, bacteriochlorophylls (Bchl), and carotenoids. Using the LH2 complex as a starting point, two chimeric LH complexes were constructed incorporating the alphaC-terminal domain of either the Rb. sphaeroides LH1 complex or the Rhodospirillum molischianum LH2 complex. The LH1 domain swap produced a new red-shifted component that comprised approximately 30% of the total absorbance. In the LH1alpha C-terminal mutant this new red-shifted species acts as the terminal emitter, with the new emission maximum located 10 nm further to the red than for the WT. Raman spectroscopy indicates that a fraction of the B850 Bchls is involved in relatively weak H-bonds, possibly involving the alphaTrp(+11) residue within the new alphaC-terminus, consistent with a more LH1-like character for one of the Bchls. The CD data indicate that the domain swaps have perturbed the native arrangement of the B850 Bchls, including the site energy difference between the alpha- and beta-bound Bchls. Thus, the normal energetic structure of the ring system has been disrupted, with one component blue shifted due to the presumed loss of an H-bond donor and the other red shifted by the influence of the new alphaC-terminal domain. The dichotomous response of the mutants to the carotenoids incorporated, spheroidenone or neurosporene, strongly suggests that the C-terminal region of the alpha polypeptide is involved in binding a carotenoid. The projection map of the LH1alpha C-terminal mutant complex was determined in negative stain at 25 A resolution, and it shows a diameter of 53 A, compared to 50 A for the WT. Hence these new spectral properties have not been accompanied by an alteration in ring size.     

Architecture of the native photosynthetic apparatus of Phaeospirillum molischianum

The ubiquity and importance of photosynthetic organisms in nature has made the molecular mechanisms of photosynthesis a widely studied subject at both structural and functional levels. A current challenge is to understand the supramolecular assembly of the proteins involved in photosynthesis in native membranes. We have used atomic force microscopy to study the architecture of the photosynthetic apparatus and analyze the structure of single molecules in chromatophores of Phaeospirillum molischianum. Core complexes are formed by the reaction center enclosed by an elliptical light harvesting complex 1. LH2 are octameric rings, assembled either with cores or in hexagonally packed LH2 antenna domains. The symmetry mismatch caused by octameric LH2 packing in a hexagonal lattice, that could be avoided in a square lattice, suggests lipophobic effects rather than specific inter-molecular interactions drive protein organization. The core and LH2 complexes are organized to form a supramolecular assembly reminiscent to that found in Rhodospirillum photometricum, and very different from that observed in Rhodobacter sphaeroides, Rb. blasticus, and Blastochloris viridis.     

Molecular architecture of photosynthetic membranes in Rhodobacter sphaeroides: the role of PufX

The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX+ and PufX- strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX- cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1beta. This arrangement, coupled with a flexible ring, would give the RC QB site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc1 complex.     

Substrate access to the active sites in aminopeptidase T, a representative of a new metallopeptidase clan

Aminopeptidase T (AmpT) from Thermus thermophilus is a metalloexopeptidase with no similarity to prototypical metallopeptidases with an HExxH or HxxEH motif. The crystal structure of the Staphylococcus aureus homologue of AmpT, which is known as aminopeptidase S (AmpS), has been reported recently. This structure revealed a dimeric protein with a very unusual, elongated shape and a large internal cavity. The active sites were found on the inner walls of the cavity and were entirely shielded from the environment, which suggested either that the dimer in the crystals was not physiologically relevant, or that an inactive conformation had been crystallized. Here, we show by gel-filtration and analytical ultracentrifugation that AmpT, like AmpS, forms dimers in solution, and we present the structure of AmpT in a crystal form with five protomers in the asymmetric unit. The five protomers take conformations that range from fully closed, as in the AmpS structure, to nearly open, so that the active site is almost directly accessible. The different conformations indicate flexibility between the AmpT N and C-domains, and explain how AmpT can be active, although the unusual AmpS dimerization mode applies to AmpT as well.     

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q(y) transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (alpha and beta) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q(y) transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 A.     

Conformational spread in a ring of proteins: a stochastic approach to allostery

We recently suggested that the sensitivity and range of a cluster of membrane receptors in bacteria would be enhanced by cooperative interactions between neighbouring proteins. Here, we examine the consequences of this "conformational spread" mechanism for an idealised one-dimensional system comprising a closed ring of identical allosteric protomers (protein molecules, or a group of protein domains operating as a unit). We show analytically and by means of Monte Carlo simulations that a ring of allosteric protomers can exhibit a switch-like response to changes in ligand concentration. We derive expressions for the sensitivity and cooperativity of switching and show that the maximum sensitivity is proportional to the number of protomers in the ring. A ring of this kind can reproduce the sensitivity and kinetics of the switch complex of a bacterial flagellar motor, for example, which is based on a ring of 34 FliM proteins. We also compare smaller rings of conformationally coupled protomers to classical allosteric proteins such as haemoglobin and show that the canonical MWC and KNF models arise naturally as limiting cases. Conformational spread appears to be a natural extension of the familiar mechanism of allostery: a physically realistic mechanism that should apply widely to many structures built from protein molecules.     

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q_y transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (α and β) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q_y transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 Å.     

High-resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2.

Light-harvesting complexes 2 (LH2) are the accessory antenna proteins in the bacterial photosynthetic apparatus and are built up of alphabeta-heterodimers containing three bacteriochlorophylls and one carotenoid each. We have used atomic force microscopy (AFM) to investigate reconstituted LH2 from Rubrivivax gelatinosus, which has a C-terminal hydrophobic extension of 21 amino acids on the alpha-subunit. High-resolution topographs revealed a nonameric organization of the regularly packed cylindrical complexes incorporated into the membrane in both orientations. Native LH2 showed one surface which protruded by approximately 6 A and one that protruded by approximately 14 A from the membrane. Topographs of samples reconstituted with thermolysin-digested LH2 revealed a height reduction of the strongly protruding surface to approximately 9 A, and a change of its surface appearance. These results suggested that the alpha-subunit of R.gelatinosus comprises a single transmembrane helix and an extrinsic C-terminus, and allowed the periplasmic surface to be assigned. Occasionally, large rings ( approximately 120 A diameter) surrounded by LH2 rings were observed. Their diameter and appearance suggest the large rings to be LH1 complexes.     

Exciton transfer between LH1 antenna complex and photosynthetic reaction center dimer

The exciton transfer between light-harvesting complex 1(LH1) and photosynthetic reaction center dimer is investigated theoretically. We assume a ring shape structure of the LH1 complex with dimer in the ring centre. The kinetic equations which describe the energy transfer between the antenna complex and reaction center dimer were derived. It was shown that the dimer does not act as a photon trap. There is a weak localization of the exciton on the dimer and there is relatively rapid back exciton transfer from dimer to antenna complex which depends on the number of the pigment molecules in the antenna ring. The relation between the rates of the exciton transfer from the antenna complex to dimer and back transfer from dimer to antenna complex has been derived.     

Statistical considerations on the formation of circular photosynthetic light-harvesting complexes from Rhodopseudomonas palustris

Depending on growth conditions, some species of purple photosynthetic bacteria contain peripheral light-harvesting (LH2) complexes that are heterogeneous owing to the presence of different protomers (containing different αβ-apoproteins). Recent spectroscopic studies of Rhodopseudomonas palustris grown under low-light conditions suggest the presence of a C ₃-symmetric LH2 nonamer comprised of two distinct protomers. The software program Cyclaplex, which enables generation and data-mining of virtual libraries of molecular rings formed upon combinatorial reactions, has been used to delineate the possible number and type of distinct nonamers as a function of numbers of distinct protomers. The yield of the C ₃-symmetric nonamer from two protomers (A and B in varying ratios) has been studied under the following conditions: (1) statistical, (2) enriched (preclusion of the B-B sequence), and (3) seeded (pre-formation of an A-B-A block). The yield of C ₃-symmetric nonamer is at most 0.98 % under statistical conditions versus 5.6 % under enriched conditions, and can be dominant under conditions of pre-seeding with an A-B-A block. In summary, the formation of any one specific nonamer even from only two protomers is unlikely on statistical grounds but must stem from enhanced free energy of formation or a directed assembly process by as-yet unknown factors.     

Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.     

Circular symmetry of the light-harvesting 1 complex from Rhodospirillum rubrum is not perturbed by interaction with the reaction center

The effect of the interaction of the reaction center (RC) upon the geometrical arrangement of the bacteriochlorophyll a (BChla) pigments in the light-harvesting 1 complex (LH1) from Rhodospirillum rubrum has been examined using single molecule spectroscopy. Fluorescence excitation spectra at 1.8 K obtained from single detergent-solubilized as well as single membrane-reconstituted LH1-RC complexes showed predominantly (>70%) a single broad absorption maximum at 880-900 nm corresponding to the Q(y) transition of the LH1 complex. This absorption band was independent of the polarization direction of the excitation light. The remaining complexes showed two mutually orthogonal absorption bands in the same wavelength region with moderate splittings in the range of DeltaE = 30-85 cm(-1). Our observations are in agreement with simulated spectra of an array of 32 strongly coupled BChla dipoles arranged in perfect circular symmetry possessing only a diagonal disorder of 相似文献   

Domain rotations between open, closed and bullet-shaped forms of the thermosome, an archaeal chaperonin

Three conformations of the thermosome, an archaeal group II chaperonin, have been determined by cryo-electron microscopy (EM). We describe an open form of the double-ring oligomer, a closed form and a bullet-shaped form with one ring open and the other closed. Domain movements have been deduced by docking atomic coordinates into the EM maps. The subunit apical domains, bearing the putative substrate binding sites, rotate about 30 degrees upwards and twist in the plane of the ring from the closed to the open conformation. The closed rings have their nucleotide binding pockets closed by the intermediate domains, but in the open rings, the pocket is accessible.     

Construction and structural analysis of tethered lipid bilayer containing photosynthetic antenna proteins for functional analysis

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAP), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.     

In silico predictions of LH2 ring sizes from the crystal structure of a single subunit using molecular dynamics simulations

Most of the currently known light‐harvesting complexes 2 (LH2) rings are formed by 8 or 9 subunits. As of now, questions like “what factors govern the LH2 ring size?” and “are there other ring sizes possible?” remain largely unanswered. Here, we investigate by means of molecular dynamics (MD) simulations and stochastic modeling the possibility of predicting the size of an LH2 ring from the sole knowledge of the high resolution crystal structure of a single subunit. Starting with single subunits of two LH2 rings with known size, that is, an 8‐ring from Rs. moliscianum (MOLI) and a 9‐ring from Rps. acidophila (ACI), and one with unknown size (referred to as X), we build atomic models of subunit dimers corresponding to assumed 8‐, 9‐, and 10‐ring geometries. After inserting each of the dimers into a lipid‐water environment, we determine the preferred angle between the corresponding subunits by three methods: (1) energy minimization, (2) free MD simulations, and (3) potential of mean force calculations. We find that the results from all three methods are consistent with each other, and when taken together, it allows one to predict with reasonable level of confidence the sizes of the corresponding ring structures. One finds that X and ACI very likely form a 9‐ring, while MOLI is more likely to form an 8‐ring than a 9‐ring. Finally, we discuss both the merits and limitations of all three prediction methods. Proteins 2011; © 2011 Wiley‐Liss, Inc.