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The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 163 Table 2. ESI-MS-determined Masses of AmHb and LtHb Polypeptide Chains and Subunits A .marina L. terrestris Globin Chains/Subunits Chain/ Subunit Mass Mass + (Da) a heme b (Da) References Chain/ Subunit Mass Mass + (Da) a heme b (Da) References Monomer (M) Monomer (M) a1 15954.4 16569.0 [83] d1 15992.4 16608.9 [23] a2 15976.8 16591.3 d2 15978.0 16594.5 b1 15920.9 16538.3 d3 15962.1 16578.6 b2 16020.1 16636.1 b 16254.4 16870.9 b3 16036.2 16652.5 c 17289.2 17905.7 c 16664.8 17280.9 a 17524.0 18140.5 d1 16983.2 17598.0 a1 19389.9 20006.4 d2 17033.1 17648.9 a2 19227.4 19843.9 a3 19065.3 19681.8 a4 18902.9 19519.4 M c 15966.7 16583.2 M c 15983.4 16599.9 Trimer (T) Trimer (T) T1 49560.4 51409.9 b1+c+d1 [83] T1 52922.6 54772.1 b+c+a1 [23] T2 49613.9 51463.4 b1+c+d2 T2 52760.0 54609.5 b+c+a2 T3 49658.6 51508.1 b2+c+d1 T3 52598.5 54448.0 b+c+a3 T4 49706.8 51556.3 b2+c+d2 T4 52435.4 54284.9 b+c+a4 T5 49724.5 51574.0 b3+c+d2 T c 49680.7 b+c+d T c 52695.2 54544.7 b+c+a Dodecamer (D) Dodecamer (D) D 204600 g [71] D 214200 g [71] 200779 e M9T= [(3a1)3 (3a2)9T] 203943 e M9T 202560 e M6T2 208689 e M6T2 204340 e M3T3 213436 e M3T3= [d3(bac)3] 284578 e M4T4 284578 e M4T4f Non-globin Chains/Subunits (Linkers) Monomer (L) Monomer (L) L1 25174.1 [83] L1a 27702.4 [23] L2 26829.7 L1b 27540.8 L1 25837.5 Dimer L2 32104.3 L3 24912.9 84
164 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al. (Table 2) contd…. Chain/ Subunit Mass Mass + (Da) a heme b (Da) References Chain/ Subunit Mass Mass + (Da) a heme b (Da) References D1 50323.1 L1+L1 [83] L4a 24169.9 D2 51981.5 L1+L2 L4b 24102.3 L4c 24019.0 a-Estimated error of measurement ± 1.0Da for M and ± 4.0Da for T b-Mass(heme)= 616.5 Da c-Weighted mean based on relative intensities [23] d-Estimated error of measurement ± 5.0 Da e-Calculated mass using average, intensity weighted masses of the apo-monomer (M) and apo-trimer (T) subunits: 15 983.2 and 52 696.1 Da for LtHb and 15 966.7 and 49 680.7 Da for AmHb respectively. At each calculated mass, 12 hemes are added. f-Subassembly proposed by Riggs and collaborators for LtHb [95, 96] g-Representative measured mass of the dodecamers obtained by ESI-MS under non-denaturing conditions were determined by denaturing ESI-MS. Given these masses, the structure the most likely to explain dodecamer masses was found to be M 3 T 3 (3 monomers and 3 trimers). From this structure and using relative proportions from ESI- MS data, a random assembly model of the dodecamers can be performed. For M and T, chains are selected from the existing pool for each species with probabilities depending on their proportion in the ESI-MS spectrum, and incorporated independently of each other in the dodecamer. Calculated expected masses are 213 436 ± 319 Da and 204 342 ± 80 Da for Lumbricus and Arenicola, respectively. These are in good agreement with measured masses of 214 200 Da and 204 600 Da. The calculated expected range and the experimental mass range are both larger for Lumbricus than for Arenicola. This supports the M 3 T 3 model for these species and suggests than the broader experimental mass range for Lumbricus is due to a higher number of possible combinations for dodecamer assemblies (200 versus 140 for Arenicola). It is worthwhile to note that masses obtained by the statistical model and by simple intensity-average mass calculation are the same since the model incorporates probabilities based on intensity from ESI-MS. However the statistical approach also gives the expected range and the standard deviation which are in good agreement with experimental data and thus supports the model. Another work investigated the theoretical mass distribution of the whole assembly for Lumbricus terrestris and Riftia pachyptila HBL-Hb [32]. Considering subunit masses and proportions from ESI-MS data, mass distributions were calculated for dodecamers, for 12 dodecamers, for linkers and finally for the whole HBL-Hb structure. For the dodecamer distribution, the M 3 T 3 model was used for Lumbricus while Riftia dodecamers were built from a random number of monomers and dimers totalizing 12 globin chains. At each step additions of different subunits were considered independent from each other. Expected masses for the whole assemblies were 3 516 ± 16 kDa and 3 284 ± 10 kDa for Lumbricus and Riftia HBL-Hb, respectively. These masses were shown to be in very good agreement with the mean of known masses for Lumbricus (3 524 ± 481 kDa) and the mean of all the known polychaete HBL-Hb masses for Riftia (3 209 ± 389 kDa). MALLS values of 3 755 ± 80 kDa and 3 503 ± 13 kDa were obtained for Lumbricus and Riftia, respectively [32, 85]. The corresponding differences represent 6.8 % and 6.7 % variations. The higher heterogeneity of Lumbricus linker subunit masses compared to Riftia results in calculated variation values (ratio range/expected mass) of 30.2 % for Lumbricus and 15.9 % for Riftia, for the linker subassembly. Probabilistic approaches are thus a convenient way to reproduce theoretical assembly from subunit masses and proportions. Expected masses and variations can support a model when compared to experimental measurements and ought to be closer to biological reality since they deal with mass distribution rather than with a unique determined mass. 5. REBUILDING RESPIRATORY PIGMENT STRUC- TURE FROM EXPERIMENTAL DATA: THE EXAMPLE OF CRUSTACEAN HC In his thorough study of respiratory pigments by sedimentation velocity and sedimentation equilibrium, Svedberg examined sedimentation constant and molecular mass for various decapod crustacean Hc [104]. Two values were determined for sedimentation constants: 16.9 S and 23.4 S. The forms present in the hemolymph depended on the species. Remarkably, Svedberg had observed the two main Hc forms found in crustacean hemolymph and had accurately determined their sedimentation constant values. However, the determined masses (360 kDa and 640 kDa instead of 450 kDa and 900 kDa, respectively) were underestimated, as was the case for annelid hemoglobins. Nowadays MALLS is a rapid and convenient way to determine the mass of an unknown Hc complex and to assess the polydispersity of the different fractions. Fig. (2) presents a typical MALLS analysis displaying the calculated molecular mass versus elution volume during a SEC experiment on Rimicaris exoculata hemolymph [105]. Crustacean samples analyzed by SEC-MALLS usually exhibit one or two Hc peaks with molecular masses around 900 kDa and 450 kDa. Since the known mass of a single subunit is about 75 kDa, these masses fit unambiguously with dodecameric and hexameric forms, respectively. This is confirmed by TEM observation of hemolymph samples in which globular particles are visible either isolated (hexamers) or associated by two (dodecamers). When analyzing Hc from different decapod species, dominant forms can be observed within each group and permit to outline some evolutionary patterns [106] (see 85
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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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22 CHAPITRE 1. INTRODUCTION FIG. 1.
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24 CHAPITRE 1. INTRODUCTION le mili
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26 CHAPITRE 1. INTRODUCTION Capacit
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28 CHAPITRE 1. INTRODUCTION TAB. 1.
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30 CHAPITRE 1. INTRODUCTION 700 600
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32 CHAPITRE 1. INTRODUCTION férent
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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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