Heme proteins—Diversity in structural characteristics,function, and folding

The characteristics of heme prosthetic groups and their binding sites have been analyzed in detail in a data set of nonhomologous heme proteins. Variations in the shape, volume, and chemical composition of the binding site, in the mode of heme binding and in the number and nature of heme–protein interactions are found to result in significantly different heme environments in proteins with different functions in biology. Differences are also seen in the properties of the apo states of the proteins. The apo states of proteins that bind heme permanently in their functional form show some disorder, ranging from local unfolding in the heme binding pocket to complete unfolding to give a random coil. In contrast, proteins that bind heme transiently are fully folded in their apo and holo states, presumably allowing both apo and holo forms to remain biologically active resisting aggregation or proteolysis. The principles identified here provide a framework for the design of de novo proteins that will exhibit tight heme ligand binding and for the identification of the function of structural genomic target proteins with heme ligands. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Electron cryomicroscopic visualization of PomA/B stator units of the sodium-driven flagellar motor in liposomes

A motor protein complex of the bacterial flagellum, PomA/B from Vibrio alginolyticus, was reconstituted into liposomes and visualized by electron cryomicroscopy. PomA/B is a sodium channel, composed of two membrane proteins, PomA and PomB, and converts ion flux to the rotation of the flagellar motor. Escherichia coli and Salmonella have a homolog called MotA/B, which utilizes proton instead of sodium ion. PomB and MotB have a peptidoglycan-binding motif in their C-terminal region, and therefore PomA/B and MotA/B are regarded as the stator. Energy filtering electron cryomicroscopy enhanced the image contrast of the proteins reconstituted into liposomes and showed that two extramembrane domains with clearly different sizes stick out of the lipid bilayers on opposite sides. Image analysis combined with gold labeling and deletion of the peptidoglycan-binding motif revealed that the longer one, approximately 70 A long, is likely to correspond to the periplasmic domain, and the other, about half size, to the cytoplasmic domain.     

Ultrastructure of the flagellar apparatus in the green flagellateSpermatozopsis similis

The ultrastructure of the flagellar apparatus of the naked, biflagellate green algaSpermatozopsis similis Preisig & Melkonian has been studied in detail using an absolute configuration analysis. The two basal bodies are displaced by 350 nm in the 1/7 o'clock direction and do not overlap proximally. They are interconnected by a principal distal connecting fibre consisting of a bundle of 5–8 nm filaments and possibly two proximal striated connecting fibres. The flagellar root system is cruciate (5-2-5-2 or 4-2-4-2 system) and contains a prominent continuous system I fibre overlying the two opposite two-stranded roots. A system II fibre is absent. Pronounced structural differences have been observed in the flagellar apparatus ultrastructure at two types of flagella orientation: During backward swimming basal bodies are parallel, the distal connecting fibre is extremely contracted; during forward swimming basal bodies assume various angles (from 20° to 180°) and the connecting fibre is about five times longer compared to the contracted state. The function of the connecting fibre as a contractile organelle and the mechanism of its contraction are discussed. On the basis of the flagellar apparatus ultrastructure,Spermatozopsis similis is related toChlamydomonas-type green algae.     

Zoospore ultrastructure in species ofTrebouxia andPseudotrebouxia (Chlorophyta)

Zoospore ultrastructure (incl. flagellar apparatus) has been investigated in three species ofTrebouxia (T. glomerata, T. erici, T. pyriformis) and one species ofPseudotrebouxia (P. impressa) using an absolute configuration analysis. Zoospores in all taxa studied are nearly identical in ultrastructure and exhibit a very distinctive disposition of cell organelles: cells are naked, biflagellate and considerably flattened along the plane of flagellar beat, the single contractile vacuole is located anteriorly in the ventral region of the cell, the nucleus is anteriorly to centrally located in the dorsal region of the cell. A single dictyosome is located close to the anterior, ventral edge of the nucleus. The chloroplast occupies a posterior position in the cell and usually has an anterior profile in the left region of the cell. There are two branched mitochondria per cell or a single mitochondrial reticulum with profiles anterior to the nucleus (in the dorsal region of the cell), and posterior to the nucleus. In zoospores ofTrebouxia spp. the posterior mitochondrial profile is associated with a microbody, inP. impressa zoospores the anterior mitochondrial profiles are associated with a microbody. The zoospores contain a distinctive system of three ER-cisternae: one system links to both basal bodies and extends to the nucleus, the other two systems subtend the plasmamembrane on the left and right broad cell surfaces and extend to the posterior region of the cell. The flagellar apparatus is structurally identical to that previously described for zoospores ofFriedmannia israelensis and exhibits basal body displacement by one basal body diameter into the 11/5 o'clock direction, a non-striated distal connecting fiber, a cruciate microtubular root system lacking system I fibers and presence of a single system II fiber which connects the basal bodies with the nucleus and runs parallel to one of the ER-strands. The left flagellar roots (X-roots) are subtended by a complex set of amorphous and striated material that connects each left root with both basal bodies.—This study demonstrates the close systematic relationship between the phycobiontsTrebouxia andPseudotrebouxia and the generaFriedmannia, Pleurastrum, andMicrothamnion and supports recent classification schemes which place all these taxa into a single order separate from otherChlorophyta. Dedicated to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday.     

THE FLAGELLAR APPARATUS OF CHILOMONAS PARAMECIUM (CRYPTOPHYCEAE) AND ITS COMPARISON WITH CERTAIN ZOOFLAGELLATES1

The major components of the internal flagellar apparatus of Chilomonas paramecium Ehr. are two large microtubular roots and a striated root paralleled by three microtubules. The two microtubular roots overlap at the basal bodies. One microtubular root follows a curved path in the anterior of the cell, and the other extends straight to the posterior passing through a groove in the nucleus. The striated root extends laterally from the basal bodies. Except that it is smaller, the posteriorly directed root bears a strong resemblance to the axostyle of oxymonads. The overall arrangement and structure of the flagellar roots is similar to the pelta, axostyle and costa of trichomonads and the pelta and axostyle of oxymonads, groups of mitochondrion-less, largely parasitic or symbiotic protozoans. An affinity between cryptomonads and oxymonads or trichomonads would have many phylogenetic implications, some of which are discussed.     

COMPARATIVE CYTOLOGY AND TAXONOMY OF THE ULVAPHYCEAE. III. THE FLAGELLAR APPARATUSES OF THE ANISOGAMETES OF DERBESIA TENUISSIMA (CHLOROPHYTA)1

The components of the flagellar apparatuses of the male and female gametes of Derbesia tenuissima (De Not.) Crouan are compared with those in other swarmers of green algae. Both the male and female gametes were found to have a cruciate microtubular root system, a non-striated capping plate which connects basal bodies, two electron dense terminal caps which partially cover the proximal end of the basal bodies, and two small system II fibrous roots. Similarities exist between these components and those suggested to be typical of ulvalean swarmers. Based upon these similarities, it is proposed that the Caulerpales be classified in the Ulvaphyceae rather than in the Charophyceae or Chlorophyceae.     

Sulcochrysis biplastida gen. et sp. nov.: Cell structure and absolute configuration of the flagellar apparatus of an enigmatic chromophyte alga

Sulcochrysis biplastida gen. et sp. nov., a golden, marine, mixotrophic flagellate is described. Cells resemble Ochromonas in light microscopic features, but they are distinct at the electron microscopic level from Ochromonas or any other typical chrysophyte. Ultrastructural features that discriminate Sulcochrysis from the Chrysophyceae are: (i) a proximal helix in the flagellar transition region; (ii) basal bodies situated in the anterior depression of the nucleus; (iii) the lack of the rhizoplast; and (iv) simple flagellar hairs lacking lateral filaments. These features suggest that Sulcochrysis is a relative of the Pedinellophyceae, Dictyochophyceae and Pelagophyceae. However, Sulcochrysis has a flagellar root system similar to that of the Ochromonas-type cell and it may use the R3 root for prey capture, as do ochromonadalean algae. The R3 root and the phagotrophic mechanism using the R3 root are interpreted as a plesiomorphy, because these are also distributed in primitive heterokonts such as the bicosoecids. Sulcochrysis has one microtubule probably homologous with the x-fibre of Bicosoeca maris Picken. Based on these features it is suggested that Sulcochrysis is an organism that links bicosoecids (Bicosoeco-phyceae), Pedinellophyceae, Dictyochophyceae and Pelagophyceae.     

FLAGELLAR HAIRS IN PRASINOPHYTES (CHLOROPHYTA): ULTRASTRUCTURE AND DISTRIBUTION ON THE FLAGELLAR SURFACE1

The flagellar hair ultrastructure of 16 strains of species of the prasinophycean genera Mantoniella, Mamiella, Pseudoscourfieldia, Nephroselmis, Tetraselmis, Scherffelia, Pterosperma, and Pyraminonas was examined in detail by whole-mount electron microscopy. The flagellar hairs of all genera displayed a high degree of ultrastructural complexity that was completely conserved within each strain. In all strains, flagellar hairs occurred on the sides of the flagella (lateral hairs); in several strains, special flagellar hairs also were found on the flagellar tips (tip hairs; absent in the Chlorodendrales and in Nephroselmis). Two groups of lateral hairs were distinguished: 1) T-hairs (“Tetraselmis-type” flagellar hairs), characterized by a smooth, tubular shaft of ca. 15 nm diameter and an overall length of 0.5–1.3 μm, and 2) P_t-hairs (“Pterosperma-type lateral flagellar hairs”), which were considerably longer (ca. 1.5–5.4 μm), characterized by a thick shaft of ca. 30 nm diameter, which was covered with a layer of regularly spaced small particles of ca. 10 nm diameter. In both groups of flagellar hairs, a strain-specific number of subunits (1–101) in linear arrangement was attached to the distal end of the shaft. Tip hairs were either structurally related to T-hairs (Mamiellales, Pseudoscourfieldia) or represented a separate group, P_t-hairs (“Pterosperma-type flagellar tip hairs”; Pterosperma, Pyramimonas). In four genera (Mantoniella, Mamiella, Pseudoscourfieldia, Nephroselmis), both groups of lateral hairs occurred together on the same cell. Interestingly in these taxa the P_t-hairs were exclusively attached to the shorter immature flagella (no. 2), but, in contrast, in Mantoniella and Pseudoscourfieldia the tip hairs were restricted to the longer mature flagellum (no. 1). Thus, flagella of different developmental status differ in their hair-scale complement. The occurrence, distribution, and ultrastructure of flagellar hairs can be used to identify and classify prasinophytes at all taxonomic levels.     

THE BASAL APPARATUS OF THE QUADRIFLAGELLATE SPERMATOZOPSIS EXSULTANS(CHLOROPHYCEAE):NUMBERING OF BASAL BODY TRIPLETS REVEALS TRIPLET INDIVIDUALITY AND DEVELOPMENTAL MODIFICATIONS1

Though there are no detectable structural differences between each of the axonemal doublets of Spermatozopsis exsultans Korshikov, basal body triplets do show structural peculiarities: one triplet consistently has an electron-dense patch appressed to its proximal part. This triplet is labeled No. 1, and all triplets are numbered in accordance to the numbering system used for other flagellate green algae. We present a detailed analysis of the basal apparatus (basal bodies and attached cytoskeletal elements) of S. exsultans and describe how basal apparatus elements are attached, very specifically, to particular basal body triplets. The analysis includes immunogold detection of centrin-containing structures and characterization of their sites of attachment to basal bodies. The sequence of basal body development in S. exsultans is deduced from what we know of other green algae. With this, we describe how the cytoskeletal structures associated with the separate basal bodies, particularly those attached to the right side of a basal body, undergo apparent morphological modifications from cell generation to generation. The data indicate that basal body triplets are truly different from one another and that this subsequent basal body asymmetry, combined with the developmental differences between basal bodies themselves, presumably accounts for the heterogeneity in the basal apparatus and any asymmetry in the cell as a whole.     

STRUCTURE AND PHYSIOLOGY OF THE HAPTONEMA IN CHRYSOCHROMULINA (PRYMNESIOPHYCEAE). I. FINE STRUCTURE OF THE FLAGELLAR/HAPTONEMATAL ROOT SYSTEM IN C. ACANTHA AND C. SIMPLEX1

The intracellular structural relationships between the flagella and haptonema in Chrysochromulina acantha Leadbeater & Manton (Prymnesiophyceae) were studied in detail and a reconstruction is presented. Three micro-tubular roots are associated with the flagellar apparatus. The largest, consisting of a sheet of approximately 20 microtubules, has its origins at the base of the left basal body. The main body of microtubules passes over the surface of a mitochondrion toward the left chloroplast and apparently terminates at a pair of microtubules oriented perpendicularly to it. Four microtubules diverge from the sheet and pass behind the left basal body. Two other roots–one consisting of a 2 + 2 + 1 arrangement of microtubules, the other of a single microtubule only—are associated with the right basal body. The two basal bodies are connected by distal and proximal fibers, and they are linked also to the base of the haptonema, three fibers extending from the haptonemal base to the right basal body, one only to the left. An additional fiber extending from the right basal body passes between the left basal body and the base of the haptonema, terminating at the largest microtubular root. Lateral extensions link this fiber to both the left basal body and the haptonematal base. Negative staining of isolated root systems of C. simplex Estep et al. shows that the arrangement of microtubules and fibrous connections is similar to that in C. acantha. The root system of C. acantha is compared to those of other members of the Prymnesiophyceae.