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The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 157 Fig. (1). Experimental data and models for AmHb and LtHb. (a) The bracelet model of HBL-Hb. Left: Electron micrographs of HBL-Hb, negatively stained. The arrows indicate the two frequent orientations; top view (t) and side view (s). Right: The bracelet model where 12 dodecamers decorate a framework of linkers. (b) Surface representation at a threshold displaying the expected molecular volume (EMV) of the native AmHb (left) and LtHb (right).The molecules observed along their 6-fold axis are displayed in such a way that the upper hexagonal layers have exactly the same orientations with the broken line joining opposite vertices vertically oriented. The vertices of the lower layer are 1° clockwise and 16° anticlockwise rotated relatively to those of the upper layer in AmHb and LtHb respectively. Another consequence of this rotation is the different shape of the holes (bordered by a black line). Because of the eclipsed disposition of the two layers, the hole is large in AmHb while the 16° rotation strongly reduces its size in LtHb (reprinted from [91], with permission from Elsevier). (c) The molecular weight of AmHb and LtHb determined by SEC-MALLS. Left: MALLS analysis of AmHb give an average molecular mass of 3 648 ± 24 kDa with a slight polydispersity as indicated by the slope of the individual estimates. Right: The calculated apparent molecular weight of the CO-hemoglobin of L. terrestris across the chromatographic peak (reprinted from [96], with permission from ASBMB). The calculated weight-average molecular mass for the entire peak is 4.10 ± 0.1 MDa 78
158 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al. (Legend Fig. 1) contd…. on the basis of the measured dn/dc value of 0.185 ml/g. (d) ESI m/z spectrum of the ~200 kDa subassembly from AmHb (left) and LtHb (right). Inset, result of deconvoluting the ESI spectrum over the m/z range 5700-7500 by MaxEnt. M, T and D set for the monomer chain, the trimer and dodecamers subassembly respectively. The number after the colon indicates the number of charges on the ion. (reprinted from [71], with permission from ASBMB). (e) MaxEnt-processed spectra of native AmHb (left) and LtHb (right) analyzed under denaturing conditions. a1-2, b, c, d1-3 design the monomeric chains, T1-4; the trimer subunits and L1-4; the linkers monomer and D1-2; the dimeric linkers (reprinted from [83] and [23], with permission from Elsevier for [23]). (f) Structural model proposed for AmHb (left) and LtHb (right). Drawing of typical HBL-Hb on top view displaying the subunits for half a Hb molecule. Left: Each AmHb one-twelfth is constituted of one trimer (T3, T4 or T5), with nine monomeric chains; M 9 T. The one-twelfth is detailed showing the possible arrangement of polypeptide chains [83]. Right: Each LtHb one-twelfth is constituted of three trimers T, with three monomeric chains; M 3 T 3 . (g) 3D reconstruction from crystallographic data. Left: The 6.2 Å electron density of AmHb. Density corresponding to the globin chains is depicted dark violet, the linker chains associated with the top of the molecule are shown in orange, while those associated with the bottom linker subunits are shown in blue (reprinted from [81], with permission from Elsevier). Right: The 5.5 Å electron density of LtHb. Density corresponding to the globin chains is depicted magenta, the linker chains associated with the top of the molecule are shown in yellow, while those associated with the bottom linker subunits are shown in blue (reprinted from [80], copyright PNAS). ied HBL-Hbs of the oligochaete Lumbricus terrestris (LtHb) and the polychaete Arenicola marina (AmHb). Prior to the use of ESI-MS and MALLS, the bracelet model was the first model proposed for HBL-Hb based on observation of dissociation products of LtHb. It consists of 12 dodecamers decorating a bracelet or framework of 30-40 linkers [93, 94] (Fig. 1a). On the basis of the mass provided by STEM analysis (3.55 x 10 6 Da) and subunit masses estimated by SDS-PAGE, the authors were able to propose a structural model where the 12 dodecamers (2.13 x 10 5 x 12 = 2.56 x 10 6 Da) account for 72 % of the total mass, in excellent agreement with the iron and heme content data [37]. Another proposed model suggested that LtHb was comprised of 192 globin subunits and 24 linkers. This model was based on native mass from light scattering measurement (4.1 x 10 6 Da), subunit masses from mass spectrometry and linker proportion from capillary electrophoresis and HPLC experiments [22, 95, 96]. Concurrent ESI-MS investigations of LtHb [23] and AmHb [83] were led to obtain the molecular masses of their constituent chains and subunits as well as their relative proportions. Fig. (1d) shows the raw ESI-MS spectra of AmHb and LtHb respectively obtained under non-denaturing conditions and Fig. (1e) shows the resulting deconvolution on a zero charge mass scale, using the MaxEnt analysis of the ESI-MS spectra under denaturing conditions. The mass spectra under denaturing conditions of the native HBL-Hb in combination with those of reduced, reduced and carbamidomethylated and unreduced carbamidomethylated forms permit the determination of the total number of Cys residues as well as the number of free and disulfide bound Cys residues. ESI-MS has also provided unambiguous determination of the nature of glycosylation, either of globin or of linker subunits [69]. This detailed mass information coupled with the MALLS determination of the molecular mass of the whole HBL-Hb complexes has allowed to propose suitable models for the quaternary structures with much greater confidence than was possible heretofore [23, 82-85] (Fig. 1f). Table 2 sums up representative masses and their SD of polypeptide chains and the globin and linker subassemblies observed in the native HBL-Hb of A. marina and L. terrestris and the closest matching masses calculated for a dodecamers globin subassembly and other possible combinations of globin subunits. Intensity-weighted mean masses for the globin subunits were derived from these data and used to generate the calculated subassembly masses in Table 2. When allowance was made for variations in the relative intensity measurements, the estimated errors in the calculated subassembly masses shown in Table 2 were < 0.05 %. It should be noted that among the oligochaete and polychaete Hb that have been investigated, there are two different patterns of globin subunits [97, 98] as well as linker subunits (see Table 1 and below). They are composed of one or more ~17 kDa monomer globin chains (M) and one or more disulfide-bonded ~50 kDa trimer subunits (T) [83, 84, 88, 99] and the linker chains are either present as monomers or disulfidebound dimers. This diversity in the small globin subunits can obviously lead to more than one combination of these subunits to give a dodecamer subassembly. LtHb contains four globin chains (chains a-d) ranging in mass from 15962 Da to 19390 Da, with the monomer subunit (chain d) existing as three isoforms (d1-d3), and chain a occurring as four glycosylated isoforms (a1-a4); the latter forms four different disulfide-bound trimer subunits with chains b and c [23]. Four monomeric linkers (L1-L4) are observed (Table 2). AmHb contains eight different globin chains (a1,a2,b1,b2,b3,c,d1 and d2) ranging in mass from 15 922 to 17 032 Da, with the b, c and d chains forming five of the six possible disulfide-bound trimers (T1-T5) (c+b1+d1, c+b1+d2, c+b1+d2, c+b2+d1, c+b2+d2, and c+b3+d2) [83]. The two linkers (L1-L2) are disulfide-bound to form hetero- or homo-dimers as detailed in Table 2. The complete ESI-MS determination of the masses of the constituent globin and linker chains and the disulfide-bound trimer subunit of LtHb provided a calculated mass of 213.434 kDa for the assembly [M] 3 [T] 3 [23] (Fig. 1d, Table 2). Furthermore, the calculated masses for the Hb comprised of 12 subassemblies and 36 or 42 linker chains are 3.523 and 3.687 MDa, respectively, in good agreement with the masses determined by scanning transmission electron microscopy and sedimentation equilibrium (3.56 ± 0.13 and 3.41 ± 0.39 MDa, respectively) [23]. However, the structural model of the AmHb dodecamers proposed by Zal and collaborators [83] (Fig. 1f) is slightly different than the model proposed by Green and collaborators: D=M 3 T 3 . A model of the quarternary structure of Arenicola marina HBL-Hb has been proposed by Zal and collaborators based on ESI-MS analysis and MALLS measurements (Fig. 1f). Three and six copies of 79
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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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26 CHAPITRE 1. INTRODUCTION Capacit
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128 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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