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70 CHAPITRE 2. ESI-MS ET MALLS APPLIQUÉS AUX PIGMENTS RESPIRATOIRES les masses des sous-unités mesurées en ESI-MS dénaturante pour établir des modèles structuraux. De plus, le MALLS peut donner des indications sur la forme des particules étudiées (R g et R h ). 2.3 Article de revue : The Structural Analysis of Large Noncovalent Oxygen Binding Proteins by MALLS and ESI-MS : A Review on Annelid Hexagonal Bilayer Hemoglobin and Crustacean Hemocyanin Les travaux réalisés sur les hémoglobines géantes d’Annélides et sur les hémocyanines de Crustacés en utilisant ces deux techniques ont été revus dans un article rédigé pendant la thèse ; des données personnelles concernant les Hcs figurent également dans l’article. Cet article est reproduit ici tel qu’il est paru ; sa bibliographie n’a pas été reprise dans la bibliographie générale de la thèse.
150 Current Protein and Peptide Science, 2008, 9, 150-180 The Structural Analysis of Large Noncovalent Oxygen Binding Proteins by MALLS and ESI-MS: A Review on Annelid Hexagonal Bilayer Hemoglobin and Crustacean Hemocyanin Matthieu Bruneaux 1,2 , Morgane Rousselot 1,2 , Emmanuelle Leize 3 , François H. Lallier 1,2 and Franck Zal 1,2,* 1 UPMC Univ. Paris 06, UMR 7144, Equipe Ecophysiologie: Adaptation et Evolution Moléculaires, Station Biologique de Roscoff, 29682, France; 2 CNRS, UMR 7144, Station Biologique de Roscoff, 29682, France; 3 CNRS-ULP, UMR 7177, Laboratoire de Dynamique et Structure Moléculaire par Spectrométrie de Masse, Institut de Chimie, ISIS, 67083 Strasbourg, France Abstract: Understanding the function of macromolecular complexes is related to a precise knowledge of their structure. These large complexes are often fragile high molecular mass noncovalent multimeric proteins. Classical biochemical methods for determination of their native mass and subunit composition were used to resolve their quaternary structure, sometimes leading to different models. Recently, the development of mass spectrometry and multi-angle laser light scattering (MALLS) has enabled absolute determination of native masses and subunit masses. Electrospray ionization mass spectrometry (ESI-MS) was used in denaturing and native conditions to probe subunit composition and noncovalent assemblies masses up to 2.25 MDa. In a complementary way, MALLS provides mass and size estimation in various aqueous solvents. ESI-MS method can also give insights into post-translational modifications (glycosylation, disulfide bridges …). By combining native mass and subunit composition data, structural models can be proposed for large edifices such as annelid extracellular hexagonal bilayer hemoglobins (HBL-Hb) and crustacean hemocyanins (Hc). Association/dissociation mechanisms, protein-protein interactions, structural diversity among species and environmental adaptations can also be addressed with these methods. With their absolute mass determination, the very high precision of spectrometry and the versatile nature of light scattering, ESI-MS and MALLS have provided a wealth of data helping to resolve parts of controversies for HBL-Hb models and opening access to new fields of investigation in structural diversity and molecular adaptation. In this review we will focus on annelid HBL-Hb and on crustacean Hc and on the original contributions of ESI- MS and MALLS in this field. Keywords: Hemoglobin, hemocyanin, mass spectrometry, light scattering, noncovalent macromolecular complexes, structural model, quaternary structure. INTRODUCTION Many biological functions necessary for cellular life are performed by multimeric protein complexes. Their constituents can interact noncovalently through electrostatic forces, hydrogen bonds, van der Waals forces or hydrophobic forces or be covalently linked by disulfide bonds. These complexes are either homo- or hetero-polymers. Each subunit can exhibit an independent activity without any particular properties emerging from the association step; on the contrary, some subunits may depend on the interactions within the complex to gain full activity. To understand the biochemical mechanisms undertaken by multimeric proteins, the need to elucidate the subunits identity and arrangement (quaternary structure) as well as their biochemical properties arises. Once able to determine structures and resulting functions, and by relating them to in vivo physico-chemical parameters and individual life history and environment, one can expect to lead an integrated physiological approach and to gain insight into physiological adaptations at the molecular level. *Address correspondence to this author at the UPMC Univ. Paris 06, UMR 7144, Equipe Ecophysiologie: Adaptation et Evolution Moléculaires, Station Biologique de Roscoff, 29682, France; Tel: 0033 (0)298292309; Fax: 0033 (0)298292324; E-mail: zal@sb-roscoff.fr The study of respiratory adaptations in animals is a pertinent field for leading such an integrated physiological approach. Respiration is essential for animal life and many animal phyla have evolved specialized transport molecules in their circulating fluid permitting enhanced oxygen delivery to the demanding tissues. These oxygen binding proteins or respiratory pigments, so-called because of their various colors when oxygenated, exhibit high structural diversity among the different groups [1-4]. Biochemical study of the multimeric respiratory pigments calls for methods able to resolve quaternary structure of the molecules and identification of the constituting subunits, as well as characterization of their interactions. When investigating these complexes, the ideal analytical technique must be able to preserve these often fragile interactions. Methods widely used for determination of the native mass are sedimentation velocity (SV), sedimentation equilibrium (SE), scanning transmission electron microscopy (STEM) and size exclusion chromatography (SEC). More recently, supramolecular mass spectrometry (MS) and multiangle laser light scattering (MALLS) were also used to ob- 1389-2037/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd. 71
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(a) FIG. 1.6 - Fonctionnement d’u
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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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