Hexagonal Bilayer Research Articles

On the molecular mass of Lumbricus erythrocruorin

A critical examination of the published molecular mass of erythrocruorin (Ec) from Lumbricus and related earthworm species reveals that the results do cluster, not at one, but at two values of the molecular mass. One cluster corresponds to ∼3.6 MDa as predicted from the Vinogradov model for the hexagonal bilayer (HBL) assembly of Lumbricus terrestris EC and as estimated from the crystal structure of HBL at 5.5 A resolution. The other cluster corresponds to ∼4.4 MDa. However, in contrast to the controversy over the molecular mass, there is a consensus that the sedimentation coefficient of intact L. terrestris Ec is ∼60S. Drawing on the occurrence of central subunits in Ec of Oenone fulgida and few other annelid species, we propose for the 4.4 MDa molecule a model of HBL supplemented by a central mass. The proposed model abides by D 6 symmetry and is a suitable candidate to represent 60S Lumbricus terrestris Ec.

The stoichiometry of the four linker subunits of Lumbricus terrestris hemoglobin suggests an asymmetric distribution

The extracellular, giant (∼3.6 MDa) hexagonal bilayer hemoglobin of the earthworm Lumbricus terrestris consists of 12 dodecamers of globin chains tethered to a central complex of 36 non-globin, linker chains (24–32 kDa). Four types of linker chains L1–L4 have been detected by electrospray ionization (ESI) and by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and isolated by reversed phase high pressure liquid chromatography (HPLC). Deconvolution of the HPLC elution profile and of the MS spectra provided the following individual linker contents, expressed as percent of the sum of the four linker peak areas: HPLC-21% L1, 37% L2, 23% L3 and 19% L4, MALDI-47% L1, 29% L2, 16% L3 and 8% L4; ESI-24% L1, 16% L2, 40% L3 and 20% L4; respectively. Comparison with electrophoretic results revealed a surprising lack of overall agreement between all the methods. The calculated mean values of the available linker contents were found to be 32±12% L1, 28±9% L2, 27±10% L3 and 13±7% L4, suggesting the following relative stoichiometry: L1: L2: L3: L4 ≈1: 1: 1: 0.5. With a total of 36 linkers, a hexagonally symmetric distribution of each of the four linker chains is impossible. Thus, the asymmetric linker distribution provides an explanation for the existence of a large dipole moment of Lumbricus terrestris hemoglobin, 17300±2300 Da ( Takashima et al., 1999).

Nested Allosteric Interactions in Extracellular Hemoglobin of the Leech Macrobdella decora

Hemoglobin from the leech Macrobdella decora belongs to the class of giant extracellular hexagonal bilayer globin structures found in annelid and vestimentiferan worms. These complexes consist of 144 heme-bearing subunits, exhibit a characteristic quaternary structure (2 x (6 x (3 x 4))), and contain tetramers as basic substructures that express cooperative oxygen binding and thus provide a structural basis for a hierarchy in allosteric interactions. A thorough analysis of the isolated tetramer indicates that it functions as a trimer of cooperatively interacting subunits and a non-cooperative monomer rather than as four interacting subunits. A thermodynamic analysis of the whole molecule favors the application of a nested Monod-Wyman-Changeux model with six cooperatively interacting 12-mer allosteric units. In contrast to the isolated tetramers, all subunits of the tetramers seem to be coupled cooperatively within the oligomerized 144-mer. Thus, besides hemocyanins and GroEL, the hexagonal bilayer hemoglobins represent another class of proteins in which the hierarchical quaternary structure provides the basis for nested interaction in their functional properties.

Mass distributions of a macromolecular assembly based on electrospray ionization mass spectrometric masses of the constituent subunits.

Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies do not have a unique mass, but rather a mass distribution. The giant extracellular erythrocruorins (Ers), approximately 3.5 MDa, comprised of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions for Lumbricus and Riftia assemblies and their globin and linker subassemblies, based on the Lumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses of Lumbricus and Riftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of approximately 9% due to the breadth of the mass distributions. The Lumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 +/- 0.481 MDa.

Surface Adsorption and Trapping of Xe on Hexagonal Ice at 180 K by Molecular Dynamics Simulations

Classical molecular dynamics (MD) simulations of Xe on the basal (0001) face of hexagonal ice at 180 K have been performed in order to investigate the mechanism of adsorption and the initial stage of absorption of a van der Waals particle into crystalline ice. The potential of mean force (PMF) as a function of the relative position of the Xe atom perpendicular to the ice surface is found to increase abruptly as the particle propagates from the disordered outermost region to the deeper hexagonally ordered region. A set of local minima observed in the PMF appears to correspond directly to the layer-by-layer crystal structure of hexagonal ice. Along with the unbound state, the first two minima (with similar free energies) that correspond to the accommodation of the guest particle on and inside the disordered outermost bilayer, respectively, are found to be populated during the MD runs. The multiple transitions of the system among these three states are also observed in accord with the PMF profile, which also suggests that the penetration of any ordered hexagonal bilayer by the Xe atom is unlikely. Furthermore, MD simulations of pristine ice (i.e., without Xe) over a 3-ns simulation period show that the initially perfect-ordered hexagonal crystalline structure of the outermost bilayer undergoes transformation to a noncrystalline phase, in which fragmented domains with hexagonal ordering persist. Moreover, the accommodation of the Xe atom inside the outermost bilayer could facilitate further disordering of the hexagonal structure of this bilayer with the formation of a completely disordered Xe−ice surface phase.

Specifics of freezing of Lennard-Jones fluid confined to molecularly thin layers

Freezing of a Lennard-Jones fluid between solid surfaces was studied using grand canonical Monte Carlo and molecular dynamics simulations. We explored the formation of frozen phases of hexagonal and orthorhombic symmetry in mono-, bi-, and tri-layer structures. The freezing transition, the type of lattice, and translational and orientational ordering were identified on the basis of orientational order parameters, in-plane two-body and three-body translational correlation functions, orientational correlation functions, and analysis of molecular mobilities. We have found that the freezing temperature is a nonmonotonous function of the pore width: orthorhombic bi-layer freezes at lower temperatures than hexagonal monolayer and hexagonal bi-layer. As the pore width increases, the transition from a hexagonal monolayer to an orthorhombic bi-layer occurred via disordered liquidlike and quasi-long-range four-fold ordered bi-layers. The latter, “quadratic” structure is characterized by an algebraically decaying four-fold orientational correlation function. The transition from crystalline hexagonal bi-layer to orthorhombic tri-layer occurs through a bi-layer structure with two uncoupled hexagonal monolayers. The quadratic phase was observed also as an intermediate structure during freezing of a liquidlike bi-layer into an orthorhombic quasi-crystal. The formation of the quadratic phase was associated with step-wise densification of fluid, a sharp increase of the local orientational order parameter, and a significant reduction of fluid mobility. In the process of solidification, the system passed through a sequence of high-density jammed structures, in which the four-fold symmetry developed progressively, as the temperature decreased.

Globin and linker sequences of the giant extracellular hemoglobin from the leech Macrobdella decora.

A detailed electrospray ionization mass spectrometric study of the approximately 3.5-MDa hexagonal bilayer hemoglobin (HBL Hb) from the pond leech Macrobdella decora has shown it to consist of at least six approximately 17-kDa globin chains, of which two are monomeric and the remaining four occur as disulfide-bonded heterodimers, and three approximately 24-kDa nonglobin linker chains (Weber et al., J. Mol. Biol. 251: 703-720, 1995). The cDNA sequences of the five major constituent chains, globin chains IIA, IIB, B, and C and linker chain L1, are reported here. The globins and linkers share 30%-50% and 20%-30% identity, respectively, with other annelid sequences. Furthermore, IIB and C align with strain A of annelid sequences, whereas IIA and B align with the strain B sequences. Although chains B and C are monomeric, chains IIA and IIB form the main disulfide-bonded dimer. They also have some unusual features: the distal His (E7) is replaced by Phe in IIA, and the highly conserved CD1Phe is replaced by Leu in IIB. In spite of these unusual features, the functional properties of Macrobdella Hb are comparable to those of other HBL Hbs. A phylogenetic analysis of the globin sequences from Macrobdella, the polychaete Tylorrhynchus, the oligochaete Lumbricus, and the vestimentiferan Lamellibrachia, indicates that the two strains originated by gene duplication followed by additional duplication of each of the two strains. The mutation rate of the linkers appeared to be faster than that of the globin chains. The phylogenetic trees constructed using the Maximum Likelihood, Neighbor-Joining and Fitch methods showed the Macrobdella globin sequences to be closest to Lumbricus, in agreement with a view of annelid evolution in which the divergence of the polychaetes occurred before the divergence of the leeches from oligochaetes.

Towards a resolution of the long-standing controversy regarding the molecular mass of extracellular erythrocruorin of the earthworm Lumbricus terrestris

The published molecular mass of erythrocruorin of Lumbricus terrestris and related earthworm species covers a bewildering range of 3.23–4.5 MDa. A critical reexamination reveals that some mass determinations were underestimated and the results do cluster, not at one, but at two values of the molecular mass. One cluster corresponds to ∼3.6 MDa, as predicted for a stoichiometry of 144 globin and 36 linker chains—the Vinogradov model for the hexagonal bilayer (HBL) assembly of Lumbricus erythrocruorin—and as estimated from the crystal structure of HBL at 5.5 Å resolution [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 7107]. The other cluster corresponds to ∼4.4 MDa. In addition, a molecular mass of 4.1 MDa, determined by multiangle laser light scattering (MALLS), stands apart of the two clusters, separated from the masses obtained by other methods of molecular mass determination. We propose a stoichiometry of 192 globin and 36 linker chains for the 4.4-MDa molecule. The 36 linkers and 144 out of 192 globin chains are identified with the HBL and the remaining 48 globins are allotted equally to the two halves of the axial cavity above and below the central torus of the structure. The proposed model is supported by the occurrence in some annelid species of erythrocruorin with centrally placed subunits [Biochim. Biophys. Acta 359 (1974) 210], and by the oxidation-dependent shedding of subunits in Lumbricus erythrocruorin. We propose further that the 4.1 MDa determination represents the weight average molecular mass of a population of molecules resulting from a partial dissociation of 4.4-MDa erythrocruorin. This interpretation seems reasonable on the background of the very low protein concentrations (∼100 μg/ml and lower) prevailing at the MALLS experiment.

Electron induced restructuring of crystalline ice adsorbed on Pt(1 1 1)

Water adsorbs on Pt(1 1 1) at 137 K to form a series of ordered hexagonal bilayer structures. At submonolayer coverages the ice coalesces to form islands with an ( 37 × 37 ) R25.3° structure which are relatively stable to low energy electrons. At saturation the overlayer compresses to form a ( 39 × 39 ) R16.1° overlayer. Further water condenses on this template to grow multilayers which become metastable with respect to an incommensurate bulk ice film. The √39 structure is unstable to electron exposure, a monolayer film restructuring to form islands of incommensurate bulk ice and exposing bare Pt. As the ice film becomes thicker, the lateral stress increases and relaxation becomes fast. Workfunction measurements show no evidence for any preferential alignment of the water dipole in the epitaxial phases, as compared to bulk ice, indicating that the films are proton disordered. Stress in the epitaxial overlayer is due to the lateral compression which maximises the binding of the first layer to Pt, rather than an ordering of the water dipoles.

Evolution of the sulfide-binding function within the globin multigenic family of the deep-sea hydrothermal vent tubeworm Riftia pachyptila.

The giant extracellular hexagonal bilayer hemoglobin (HBL-Hb) of the deep-sea hydrothermal vent tube worm Riftia pachyptila is able to transport simultaneously O(2) and H(2)S in the blood from the gills to a specific organ: the trophosome that harbors sulfide-oxidizing endosymbionts. This vascular HBL-Hb is made of 144 globins from which four globin types (A1, A2, B1, and B2) coevolve. The H(2)S is bound at a specific location (not on the heme site) onto two of these globin types. In order to understand how such a function emerged and evolved in vestimentiferans and other related annelids, six partial cDNAs corresponding to the six globins known to compose the multigenic family of R. pachyptila have been identified and sequenced. These partial sequences (ca. 120 amino acids, i.e., 80% of the entire protein) were used to reconstruct molecular phylogenies in order to trace duplication events that have led to the family organization of these globins and to locate the position of the free cysteine residues known to bind H(2)S. From these sequences, only two free cysteine residues have been found to occur, at positions Cys + 1 (i.e., 1 a.a. from the well-conserved distal histidine) and Cys + 11 (i.e., 11 a.a. from the same histidine) in globins B2 and A2, respectively. These two positions are well conserved in annelids, vestimentiferans, and pogonophorans, which live in sulfidic environments. The structural comparison of the hydrophobic environment that surrounds these cysteine residues (the sulfide-binding domain) using hydrophobic cluster analysis plots, together with the cysteine positions in paralogous strains, suggests that the sulfide-binding function might have emerged before the annelid radiation in order to detoxify this toxic compound. Moreover, globin evolutionary rates are highly different between paralogous strains. This suggests that either the two globin subfamilies involved in the sulfide-binding function (A2 and B2) have evolved under strong directional selective constraints (negative selection) and that the two other globins (A1 and B1) have accumulated more substitutions through positive selection or have evolved neutrally after a relaxation of selection pressures. A likely scenario on the evolution of this multigenic family is proposed and discussed from this data set.

Physisorption of water on salt surfaces

Results of ab initio calculations of water adsorption on clean NaCl(1 0 0) surfaces show that favorable adsorption geometries critically depend on the competition of water–surface and water–water interactions. At the most favorable adsorption site, the oxygen is near the cation with hydrogens forming hydrogen bonds with nearest anions. For monolayer adsorption, the water–water interaction is decisive for the clustering of water molecules. We also discuss a hexagonal (2×4) bilayer ice-like structure. Molecular-dynamics simulations give new insight into the dynamic stability of different adsorption geometries. Finally, some complementary results for KCl are given, in which case the interaction of oxygen and cation is weaker.

Observation of Large, Non-covalent Globin Subassemblies in the ∼3600 kDa Hexagonal Bilayer Hemoglobins by Electrospray Ionization Time-of-Flight Mass Spectrometry

A non-covalent globin subassembly comprising 12 globin chains (204 to 214 kDa) was observed directly by electrospray ionization time-of-flight mass spectrometry in the native hexagonal bilayer hemoglobins from the oligochaetes Lumbricus terrestris and Tubifex tubifex, the polychaetes Tylorrhynchus heterochaetus, Arenicola marina, Amphitrite ornata and Alvinella pompejana, the leeches Macrobdella decora, Haemopis grandis and Nephelopsis oscura and the chlorocruorin from the polychaete Myxicola infundibulum, over the pH range 3.5-7.0. The Hb from the deep-sea polychaete Alvinella exhibited in addition, peaks at ∼107 kDa and at ∼285 kDa, which were assigned to subassemblies of six globin chains and of 12 globin chains with three non-globin linker chains, respectively. The experimental masses decreased slightly with increased de-clustering potential (60 to 160 V) and were generally 0.1 to 0.2 % higher than the calculated masses, due probably to complexation with cations and water molecules.

Transmission electron microscopical studies on some haemolymph proteins from the marine polychaete Nereis virens

The hexagonal bilayer haemoglobin molecule from Nereis virens has been investigated in a comparative study using several different negative stain electron microscopical specimen preparations (i.e. by conventional adsorption to continuous carbon support films, by the negative staining-carbon film technique and by negative staining across the holes of holey carbon support films with air-drying and rapid freezing/cryo-negative staining). The benefits and limitations of these different approaches are indicated, with the overall conclusion that negative staining with ammonium molybdate across holes creates the best possibilities for molecular imaging, and also has the potential for the creation of two-dimensional (2D) crystals/arrays at the fluid–air interface. Of the different negative staining procedures presented, cryo-negative staining reveals the greatest details of N. virens haemoglobin. This is exemplified by the direct visualisation of the central linker-assembly within the haemoglobin molecule, a structural feature less clearly defined by the other negative staining techniques. A discoidal lipoprotein molecule (diameter 30–60 nm; thickness ca 8 nm) has been detected in N. virens, which represents the first documented account of an annelid haemolymph lipoprotein. The biological implications of this lipoprotein for lipid transport remain to be established. The presence of a low concentration of ferritin molecules in N. virens haemolymph is also shown, assisted by the formation of small 2D ferritin arrays in negatively stained specimens prepared across holes.

Protein and Gene Structure of a Chlorocruorin Chain of Eudistylia vancouverii

The polychaete annelid, Eudistylia vancouverii, contains as oxygen carrier a hexagonal bilayer (HBL) chlorocruorin. One of the globin chains, chain a1, has 142 amino acids (Mr 16,054.99) and its sequence deviates strongly from other nonvertebrate globin sequences. Unprecedented, it displays a Phe at the distal position E7 as well as at position B10, creating a very hydrophobic heme pocket probably responsible for the low oxygen affinity of the native molecule. Phylogenetic analysis of annelid globin chains clearly proves that globin chain a1 belongs to type I of globin chains having a pattern of 3 cysteine residues essential for the aggregation into a HBL structure. The gene coding for globin chain a1 is interrupted by 2 introns at the conserved positions B12.2 and G7.0. Based on protein and gene structure it can therefore be concluded that the globin chains of chlorocruorins are not fundamentally different from other annelid globin chains.

Occurrence of two architectural types of hexagonal bilayer hemoglobin in annelids: comparison of 3D reconstruction volumes of Arenicola marina and Lumbricus terrestris hemoglobins

A 3D reconstruction at 25 Å resolution of native hemoglobin of the polychaete worm Arenicola marina was carried out from frozen-hydrated specimens examined in the electron microscope. The reconstruction volume of this large extracellular multimeric respiratory pigment appears as a hexagonal bilayer structure with eclipsed vertices in its upper and lower hexagonal layers. Conversely, in hemoglobins of oligochaetes, achaetes, and vestimentiferans and in chlorocruorins of the Sabellidae (polychaete) family, the vertices of the upper layer are 16° clockwise rotated with respect to those of the lower layer. The fact that two other polychaete hemoglobins ( Alvinella pompejana and Tylorrhynchus heterochaetus) have the same architecture as Arenicola led us to define two types of hexagonal bilayer hemoglobins/chlorocruorins: (i) type-I present in oligochaete, achaete, and vestimentiferan hemoglobins and in Sabellidae chlorocruorins; and (ii) type-II present in polychaete hemoglobins. A comparative study of the hemoglobins of Lumbricus terrestris (type-I) and Arenicola marina (type-II) showed that only two small differences located in the c4 and c5 linking units are responsible of the important architectural difference present in oligomers. A likely scheme proposed to explain the phylogenic distribution of the two types suggests that Clitellata, Sabellida (polychaete), and vestimentiferan hemoglobins and chlorocruorins derive from a type-I ancestral molecule, while Terebellida ( Alvinella), Phyllodocida ( Tylorrhynchus), and Scolecida ( Arenicola) and possibly other polychaetes derive from an ancestor molecule with type-II hemoglobin. The architectures of the hollow globular substructures are highly similar in Arenicola and Lumbricus hemoglobins, with 12 globin chains and three linking units (c3a, c3b, and c4). The central piece of Arenicola hemoglobin is an ellipsoid while that of Lumbricus is a toroid. No phylogenic correlation could be found between the structure of the central pieces and the architecture type.

Subunit distribution of calcium-binding sites in Lumbricus terrestris hemoglobin.

The giant, approximately 3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36-42 linkers L1-L4 (24-32 kDa). It is known to contain 50-80 Ca and 2-4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH approximately 2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca2+ and all the Cu and Zn, the globin subassembly had 0.4 to approximately 3 Ca2+, depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH approximately 2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH approximately 2.2 and at neutral pH demonstrated avid binding of 1 Ca2+ and additional weaker binding of 7 Ca2+ in the presence of added Ca2+. Based on these and previous results which document the heterogeneous nature of the Ca2+-binding sites in Lumbricus Hb, we propose three classes of Ca2+-binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) approximately 30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) approximately 25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1-100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.

Combinatorics of giant hexagonal bilayer hemoglobins

The paper discusses combinatorial and probabilistic models allowing to characterize various aspects of spacial symmetry and structural heterogeneity of the giant hexagonal bilayer hemoglobins (HBL Hb). Linker–dodecamer configurations of HBL are described for two and four linker types (occurring in the two most studied HBL Hb of Arenicola and Lumbricus, respectively), and the most probable configurations are found. It is shown that, for HBL with marked dodecamers, the number of `normal-marked' pairs of dodecamers in homological position follows a binomial distribution. The group of symmetries of the dodecamer substructure of HBL is identified with the dihedral group D 6. Under natural symmetry assumptions, the total dipole moment of the dodecamer substructure of HBL is shown to be zero. Biological implications of the mathematical findings are discussed.

Electrospray Ionization Mass Spectrometric Determination of the Molecular Mass of the ∼200-kDa Globin Dodecamer Subassemblies in Hexagonal Bilayer Hemoglobins

Hexagonal bilayer hemoglobins (Hbs) are approximately 3.6-MDa complexes of approximately 17-kDa globin chains and 24-32-kDa, nonglobin linker chains in a approximately 2:1 mass ratio found in annelids and related species. Studies of the dissociation and reassembly of Lumbricus terrestris Hb have provided ample evidence for the presence of a approximately 200-kDa linker-free subassembly consisting of monomer (M) and disulfide-bonded trimer (T) subunits. Electrospray ionization mass spectrometry (ESI-MS) of the subassemblies obtained by gel filtration of partially dissociated L. terrestris and Arenicola marina Hbs showed the presence of noncovalent complexes of M and T subunits with masses in the 213. 3-215.4 and 204.6-205.6 kDa ranges, respectively. The observed mass of the L. terrestris subassembly decreased linearly with an increase in de-clustering voltage from approximately 215,400 Da at 60 V to approximately 213,300 Da at 200 V. In contrast, the mass of the A. marina complex decreased linearly from 60 to 120 V and reached an asymptote at approximately 204,600 Da (180-200 V). The decrease in mass was probably due to the progressive removal of complexed water and alkali metal cations. ESI-MS at an acidic pH showed both subassemblies to consist of only M and T subunits, and the experimental masses demonstrated them to have the composition M(3)T(3). Because there are three isoforms of M and four isoforms of T in Lumbricus and two isoforms of M and 5 isoforms of T in Arenicola, the masses of the M(3)T(3) subassemblies are not unique. A random assembly model was used to calculate the mass distributions of the subassemblies, using the known ESI-MS masses and relative intensities of the M and T subunit isforms. The expected mass of randomly assembled subassemblies was 213,436 Da for Lumbricus Hb and 204,342 Da for Arenicola Hb, in good agreement with the experimental values.

The role of linkers in the reassembly of the 3.6 MDa hexagonal bilayer hemoglobin from lumbricus terrestris

The extent and kinetics of reassembly of the four groups of linkers L1-L4 with 213 kDa subassemblies of twelve globin chains D, (bac)3(d)3, isolated from the ∼3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris, was investigated using gel filtration. The reassembled HBL’s were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH ∼2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from ⩾13 % in the absence of Ca(II) to ⩽75 % in 1-10 mM Ca(II). Reassembly to HBL structures whose linker contents, STEM images and masses were similar to the native Hb was observed with all the linkers (⩾75 %), with ternary and binary linker combinations (40-50 %) and with individual linkers producing yields increasing in the following order: L1=1-3 %, L2≈L3=10-20 % and L4=35-55 %. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the 3-fold symmetry of D.

An electrospray ionization mass spectrometric study of the giant, extracellular, hexagonal bilayer hemoglobin of the leech Haemopis grandis provides a complete enumeration of its subunits

The complex, multiply charged electrospray ionization mass spectra of the extracellular, ∼3500 kDa, hexagonal bilayer hemoglobin from the leech Haemopis grandis and its carbamidomethylated, reduced and reduced/carbamidomethylated forms were deconvoluted using a maximum entropy approach to provide the corresponding zero-charge spectra. Three groups of peaks were observed: monomeric globin chains a1–a4 at ∼17 kDa (16 634.1, 17 013.4, 17 592.9, and 17 573.3 Da with relative intensities of 16:4:12:1, respectively), linker chains L1–L3 at ∼24 kDa (24 004.2, 24 449.2 and 24 548.3 Da, with relative intensities of 5:1:2.5, respectively) and subunits D1 and D2 at 32 501.1 and 32 629.6 Da, respectively, with equal intensities. Reduction of the native hemoglobin with dithiothreitol resulted in a decrease in the mass of linker L2 by 115.1 Da, indicating cysteinylation, the disappearance of subunits D1 and D2 and the concomitant appearance of globin chains b (16 098.8 Da), c1 (16 403.9 Da), and c2 (16 532.5 Da), suggesting that subunits D1 and D2 are disulfide-bonded dimers b + c1 and b + c2, respectively. All the globin chains appear to have one intrachain disulfide bond, and globins b, c1, and c2 have an additional Cys which forms the interchain disulfide bond in D1 and D2. The linkers L1–L3 contain 10, 9, and 10 Cys residues, respectively, all forming intrachain disulfide bonds, except for the odd residue in L2 which is proposed to be the site of cysteinylation. The relative intensities of the three groups of peaks a1 + a2 + a3 + a4:L1 + L2 + L3:D1 + D2 are 1.65:1.7:0.8 in native hemoglobin. All the subunits in a second sample evinced substantial glycosylation, with up to five hexoses per subunit. A model of the quaternary structure of Haemopis hemoglobin is proposed, consisting of 72 monomeric globins and 36 globin dimers for a total of 144 globin chains and 42 linkers with a calculated total mass of 3506 kDa.