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The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 151 tain precise and accurate masses of native molecules [5, 6]. To obtain the masses of each constituent, gel electrophoresis has been widely used but the current method of choice is mass spectrometry under denaturing conditions. By associating data concerning both the native protein and its subunits, new insights into the quaternary structure and the fine tuning of its physiological functions can be achieved. 1. INVERTEBRATE OXYGEN BINDING PROTEINS: THE EXAMPLE OF GIANT HEXAGONAL BILAYER HEMOGLOBIN AND HEMOCYANIN Oxygen binding proteins occur in all phyla but reach a high diversity in metazoans [2-4]. They exhibit a high diversity of localization and structure. They are found in tissues and in coelomic or vascular fluids. They can be stored in cells (intracellular pigments) or freely dissolved in the internal fluids (extracellular pigments). They are metalloproteins in which either iron (hemoglobin, hemerythrin) or copper (hemocyanin) reversibly bind O 2 . In hemoglobins, one iron atom is bound to the tetrapyrrole ring to form the heme group. The heme is linked to a polypeptide which presents a typical globin-fold [4]. They have been notably observed in annelids, arthropods and molluscs. They range from single chains (e.g. myoglobin, cytoglobin, leghemoglobin) found in bacteria, algae, protozoa and plants to large, multisubunit, multidomain complexes found in nematodes, molluscs and crustaceans, and the giant annelid hemoglobins comprised of globin and nonglobin subunits [4]. In hemocyanins, two copper atoms are bound to histidine residues of the polypeptide chain; the resulting “blue” proteins are present in molluscs and arthropods. In this review, we will focus on the analytical structure of two classes of multimeric respiratory pigments: the annelid extracellular hexagonal bilayer hemoglobins (HBL-Hb) and crustacean hemocyanins (Hc). These pigments exhibit high complexation levels and the wide range of living environments of the concerned animal phyla allows for different functional properties among species. In addition, the recently proposed use of Arenicola marina Hb (AmHb) as a model for a potential blood substitute [7, 8] and the use of arthropod Hc as a model for nested allostery further support the interest of structural investigation of these complexes [9-12]. 1.1. The Hexagonal Bilayer Hemoglobin (HBL-Hb) of Annelids The annelid phylum comprises over 8000 species subdivided into terrestrial oligochaetes, aquatic leeches, and marine polychaetes. Annelids are present in a wide variety of biotopes, from aerated soil (Lumbricus terrestris) to challenging hypoxic or toxic environments (intertidal muds for Arenicola marina; deep-sea hydrothermal vents for Alvinella pompejana). Most of them have extracellular or intracellular hemoglobins (Hbs) and sometimes both [13, 14]. However, in many annelids, oxygen transport relies on giant extracellular respiratory proteins (~3.6 x 10 6 Da), known as either erythrocruorins or hexagonal bilayer hemoglobins (HBL- Hbs). Such large complexes offer a number of important advantages as oxygen transport vehicles: HBL-Hbs are readily retained in the vascular system as freely dissolved entities, each complex possesses large oxygen binding capacity and subunits can be arranged to permit cooperative oxygen binding and additional regulatory features enhancing oxygen transport. The HBL-Hbs are heteromultimeric complexes consisting of globins (14-18 kDa) and nonglobin, linker chains (23-28 kDa) in an approximate 2:1 ratio, and represent a summit of complexity for heme proteins binding O 2 reversibly. Chlorocruorins (Chl) are respiratory pigments structurally similar to HBL-Hb except for a different side group in the tetrapyrrole ring. Their absorption spectrum is thus modified so that the oxygenated form is green rather than red. They are found in some annelid families (Sabellidae, Serpulidae, Chlorohaemidae, Amphateridae [4]). The HBL-Hbs of the polychaete A. marina and the oligochaete L. terrestris were among the first proteins investigated by ultracentrifugation [15] and electron microscopy [16]. They exhibit a highly symmetric hexagonal bilayer appearance (HBL), with dimensions of about 20 nm x 30 nm [17]. The first biochemical studies by Svedberg and collaborators yielded sedimentation constant for numerous erythrocruorins, ranging from 54.1 S to 61.5 S, and also produced a first estimation of the mass for A. marina and L. terrestris hemoglobins of about 2.8 MDa [15, 18]. HBL-Hbs exhibit some original and remarkable properties, such as an acidic isoelectric point, a high resistance to auto-oxidation and self-assembling properties, which could be explained by the selection of robust structures for oxygen transport as a freely dissolved molecule in the blood. The high aggregation degree also permits a broad range of cooperativity (from n 50 = 0.91 in Amphitrite ornata [19] to n 50 = 6.5 in Megascolides australis [20]). A large affinity range is also observed between species (P 50 = 0.2 Torr for Alvinella pompejana Hb at 20°C; P 50 = 80 Torr for Spirographis spallanzanii Chl at 20°C [21]). As the global structure detected by transmission electron microscopy (TEM) seems quite uniform in annelids from different biotopes, the understanding of the inner structural organization of these respiratory complexes is of crucial interest to point out potential specific molecular adaptations to the environmental conditions. The determination of the precise composition and structure of these complexes has been a major issue in invertebrate Hb biochemistry for a long time. Determination of a model called for knowledge of the complex mass, the subunit masses and stoichiometry and the spatial arrangement of the chains. Several models were proposed to account for the hexagonal bilayer structure observed for all extracellular giant hemoglobins and the relationship between globin and linker subunits. Determinations of native masses sometimes led to different results as is the case for L. terrestris [22, 23]. Availability of high resolution methods for determination of native and denatured masses was a keystone in the resolution of parts of the controversies in this field. 1.2. Arthropod Hemocyanin (Hc) Hemocyanin is the respiratory pigment responsible for dioxygen transport in many arthropods and molluscs. It is a polypeptide complex of high molecular mass, ranging from 450 kDa (crustaceans) to about 10 MDa (gastropods). They share a seemingly similar active site involving two Cu atoms 72
152 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al. linked to histidine residues to bind one O 2 molecule [3]. However, apart from the fact that Cu is the metallic atom responsible for O 2 binding in both phyla, arthropod and molluscan Hc have very different structures. Arthropod hemocyanin basic functional subunit is a ~75 kDa polypeptide chain with only one active site. Hexamer is the basic aggregation state met in the hemolymph but dodecamers and complexes of higher molecular mass can be formed from hexamer association, depending on the species. The largest known assembly stands for Limulus polyphemus with a 48-mer (~3.6 MDa) resulting from the association of 8 hexamers. The spatial arrangement of the hexamers in the whole assembly can vary from one group to another. In crustacean, Hc is usually found as hexamers and dodecamers. Many species can produce different subunits and thus different complexes can be formed. The combination of high aggregation states and basic subunit diversity leads to a very high potential structural plasticity. Such hierarchical levels of assembly enable modulation of the oxygen binding properties of the pigments. Affinity and cooperativity can be affected by various factors such as H + , Ca 2+ , Mg 2+ and other inorganic ions, organic cofactors or the aggregation state itself [24, 25]. These mechanisms allow individuals to rapidly modulate their pigment properties when facing a change in environmental conditions. The role of structural plasticity in the response to environmental challenge has also been demonstrated in some species [26-29]. Considering the aggregation properties of hemocyanins and the subunit diversity in each species, it is crucial to know which complexes are present and their precise composition in order to be able to compare between species of the same phyla and between different conditions for a unique species. 2. METHODS USUALLY EMPLOYED TO INVESTI- GATE MACROMOLECULAR COMPLEX MASS AND STRUCTURE Determination of protein mass and structure has been a central field of investigation since the beginning of biochemistry. Numerous methods have been developed along the decades to obtain information about mass, size, composition, subunit arrangement and physical or biochemical constants. Commonly used methods in this field are sedimentation velocity (SV), sedimentation equilibrium (SE), gel electrophoresis, size-exclusion chromatography (SEC), scanning electron transmission microscopy (STEM), cryoelectron microscopy (cryo-EM), crystallography and primary sequence determination. SV and SE are powerful methods for investigation of native mass, diffusion coefficient, hydrodynamic shape, protein interactions and stoichiometry of assemblies [30, 31]. However, an inaccuracy on the specific volume of the molecule can exist and results in an up to 10 % error [32]. SEC also allows determination of native masses but is very inaccurate because of the influence of the protein shape and potential interaction with the matrix [33, 34]. Both SEC and ultracentrifugation techniques can use various solvents to test their effect on protein stability. The STEM method permits determination of native mass with a 3-10 % precision while providing images of the macromolecules for visual control and local mass mapping [35]. Unfortunately very few laboratories have access to the needed instrumentation. On the contrary, gel electrophoresis methods can be relatively easily developed in a laboratory and provides rapid analysis of samples. The resolution and accuracy are low but close proteins can sometimes be separated depending on their isoelectric point in 2D gel electrophoresis. Low quantification reproducibility can also be a limitation [36]. Nonetheless the rapidity and ease of use of classical SDS-PAGE make it very convenient for rapid monitoring of protein purification for example. Protein spots can also be excised and analyzed by mass spectrometry. Crystallography or 3D reconstruction by cryoelectron microscopy [37] are useful for determining subunits spatial arrangement and interactions between them. Crystallography can yield almost all structural information (active-site structure, interaction) if the structure is well resolved but it is really difficult to obtain a crystal structure of a large biopolymer such as hexagonal-bilayer hemoglobin or hemocyanin. Polypeptide chain mass can also be calculated from cDNA sequence. However, these methods cannot reveal heterogeneous post-translational modifications. Major difficulties of crystallography are the production of pure protein and the determination of crystallization conditions. Cryo-EM can be easier to achieve but also calls for highly specialized material and yields a lower resolution. However, hybrid approaches combining cryo-EM, sequence analysis and X-ray can yield molecular model of entire complexes. All the cited techniques are of major interest for investigation of protein mass and structure: they can either provide high quality information (SE, SV, STEM, crystallography and cryo-EM) or provide rapid and convenient analysis (gel electrophoresis, SEC). Major limitations are either accuracy or precision: the resulting error for sedimentation techniques and gel electrophoresis can be as high as 10% for invertebrate respiratory pigments [32]. Another limitation is the need for a very specific instrumentation (e.g. crystallography, STEM) or the delay from the beginning of the process until achievement (e.g. crystallography, cDNA sequencing). In this context and to study high molecular mass noncovalent complexes such as invertebrate respiratory pigments, one would expect to use methods enabling relatively rapid analysis of samples, in native and denaturing conditions to obtain information about quaternary structure, with a high precision to be able to detect slight variations in close molecular species. Multi-angle laser light scattering (MALLS) and mass spectrometry are two complementary approaches permitting to satisfy most of these requirements. 3. TWO COMPLEMENTARY TECHNIQUES YIEL- DING PROTEIN MASS: LIGHT SCATTERING AND MASS SPECTROMETRY 3.1. Multi-Angle Laser Light Scattering (MALLS) Recent improvements in instrumentation have made the light scattering (LS) technique a powerful method to determine the absolute molecular mass of proteins. On-line connection of advanced laser LS devices with high-performance liquid chromatography (HPLC) equipment can provide quick and accurate molecular mass determinations for proteins and help to characterize protein-protein interactions in solution. 73
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THESE DE DOCTORAT DE L’UNIVERSITE
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i Résumé / Abstract Réponse adap
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iv SOMMAIRE 4.2 Objectifs de l’é
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2 CHAPITRE 1. INTRODUCTION Les éco
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4 CHAPITRE 1. INTRODUCTION 1.1 Mili
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6 CHAPITRE 1. INTRODUCTION Les prin
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8 CHAPITRE 1. INTRODUCTION TAB. 1.1
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10 CHAPITRE 1. INTRODUCTION et cycl
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FIG. 1.5 - Localisation des princip
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(a) FIG. 1.6 - Fonctionnement d’u
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16 CHAPITRE 1. INTRODUCTION TAB. 1.
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18 CHAPITRE 1. INTRODUCTION FIG. 1.
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122 CHAPITRE 3. STRUCTURE DE L’HC
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134 CHAPITRE 4. PLASTICITÉ PHÉNOT
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Dénaturant Natif
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172 CHAPITRE 5. ADAPTATIONS RESPIRA
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216 CHAPITRE 6. CONCLUSION ET PERSP
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Annexe A Détermination des masses
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229 10000 h280 .M h) h280−h337 80
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Annexe B Détermination des distrib
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233 Modèle 2 : 2 sous-unités lég
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235 1 0.9 0.8 6 s-u aléatoires 2 s
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Bibliographie Adair, G. S. The hemo
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BIBLIOGRAPHIE 239 Catalina, M. I.,
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BIBLIOGRAPHIE 241 Farmer, C. S., J.
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BIBLIOGRAPHIE 243 Hourdez, S. Adapt
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BIBLIOGRAPHIE 245 Mangum, C. P. Sub
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BIBLIOGRAPHIE 247 Rainer, J., C. P.
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BIBLIOGRAPHIE 249 Svedberg, T. et I
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BIBLIOGRAPHIE 251 Vanin, S., E. Neg
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Liste des figures 1.1 Cycle des mar
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LISTE DES FIGURES 255 4.17 Profils
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Table des matières Résumé Sommai
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TABLE DES MATIÈRES 261 4.3.5 Spect
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TABLE DES MATIÈRES 263 Table des m
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