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The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 165 below). Crossed immunoelectrophoresis suggested that a particular type of subunit, subunit beta, is necessary for dodecamer assembly in most species, but that a few groups have independently evolved the ability to produce dodecamers from the other subunit types [106]. The quality of the MALLS analysis depends on the efficiency of the separation process: two badly-resolved molecular species will yield an average molecular mass with high polydispersity. In addition, a limitation of the MALLS method is that complexes with the same number of subunits but of slightly different types will differ only very slightly in mass. They cannot be resolved by SEC and thus MALLS will only give an average mass for all complexes of the same order. ESI-MS can provide further characterization in this case. ESI-MS has also been used recently for investigation of crustacean Hc. Noncovalent ESI-MS can resolve different molecular species in hemolymph without any prior separation. However, the sample preparation is a critical step in the ESI-MS procedure and Hc samples are desalted thoroughly in 10 mM ammonium acetate through at least 7 cycles of concentration/dilution on centrifugal filters before analysis; an incomplete desalting causes low signal-to-noise ratio. Under the adjusted conditions of noncovalent ESI-MS [62, 73] (see before), the different species are clearly visible as several Gaussian peak distributions along the m/z axis as presented in Fig. (3a). These distributions are well separated between different aggregation states (hexamers versus dodecamers); for a given aggregation state several close distributions are sometimes detected, corresponding to a slight mass difference. In Fig. (3a), two distributions are observed for hexamer (a,b) and dodecamer (c,d). As previously noted, such discrimination is not possible using the average mass determined by MALLS because of the very close mass and coelution of these complexes. Thus ESI-MS is a complementary method enabling thorough investigation of the complex native masses. In addition to being a technique allowing high mass accuracy measurements, ESI-MS can also detect species present in low amount or hardly or not resolved by MALLS due to coelution of different complexes. In Fig. (3a) a higher aggregate is observed with a mass of 1 374 136 Da (18-meric form). Higher masses corresponding to 24-mers and even 30-mers can be also be found [62]. It is known that aggregation can occur in the ESI when sample concentration is too high but it is unlikely that the assemblies observed here could be artifacts at least for the 18-mer as it can be observed by MALLS under low flow rate (0.25 ml/min, personal observation). However, the very low detected amount of these “unusual” complexes raises the question of their biological significance ; since no study by electron microscopy was performed on them, it is still possible that they arise from random post-translational cross-linking. For such high masses, charge state assignation may be difficult due to peak enlargement and the consecutive proximity of standard deviation values calculated for subsequent charge states [62, 74]. Even the use of deconvolution programs like MaxEnt cannot resolve the problem. The precision of the determined masses decreases with increasing masses but is nonetheless sufficient to unambiguously determine the number of subunit for each distribution as the uncertainty is inferior to the subunit average mass. Fig. (2). SEC-MALLS analysis obtained for a Rimicaris exoculata hemolymph sample. Analysis was performed with a Superose 6-C column. Optical density at 280nm (full curve) and estimated molecular weight (dots) are represented as a function of the eluted volume. Insert: cumulative molecular weight curve (reprinted from [105], with permission). 86
166 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al. Fig. (3). ESI-MS analysis of Carcinus maenas Hc. (a) ESI-MS spectrum obtained in native conditions. Charge state of main peaks and magnification are indicated on the spectrum. Analysis was performed on whole, desalted hemolymph in 10mM ammonium acetate buffer, pH 6.8 (b) Deconvoluted mass spectrum obtained in denaturing conditions from whole, desalted hemolymph diluted in 50:50:1 water-acetonitrile-formic acid mixture, pH 2.3 (reprinted from [62]). Analysis of Hc with denaturing ESI-MS reveals a high subunit diversity, from 4 to 8-9 different subunits for a given species (Table 3 and Fig. 3b). All subunits have a molecular mass close to 75 kDa. Due to its high mass accuracy, ESI- MS can detect more subunits than polyacrylamide gel electrophoresis (PAGE) [107]. ESI-MS is thus the method of choice for complete investigation of subunit composition. The structural diversity observed in the decapod species which have been studied by MS indicates a narrow range of structures (hexamer assemblies comprised of ~75 kDa chains), but high diversity within this range (see below). Whereas immunochemistry permitted the identification of subunit types differing in function, methods such as PAGE and ESI-MS can help to detect additional heterogeneities which could be functionally neutral but nevertheless useful for phylogenic studies. MALLS and ESI-MS analysis produce robust data for Hc structural modeling. Hc structure is much easier to analyze than annelid HLB-Hb structure: the lower native mass allows ESI-MS analysis of the native assembly and the excellent agreement between the model, subunit masses and native masses dismiss any ambiguity. Crystallographic and cryo- EM data of native proteins confirm these models [108, 109]. ESI-MS results are very useful to investigate the heterogeneity of the Hc complexes. It has higher resolution than MALLS and is more sensitive for high order complexes in low amount. However the system is also much more expensive and requires competent and specialized users for operation. While MALLS provides masses in a physiological buffer, ESI-MS measures masses of gas-phase ions. Thus MALLS and ESI-MS are complementary methods to explore structure heterogeneity and its relevance in biological conditions. Fig. (4a) permits to compare between native masses determined by non-covalent ESI-MS and MALLS and theoretical masses calculated from subunit masses. The good agreement between the determined masses shows the reliability of the method and validates the use of ESI-MS for investigation of noncovalent assemblies representative of the native state in solution. It is worthwhile to note that MALLS gives higher masses than those predicted from subunit masses (see below). For Carcinus maenas, slightly higher differences (up to 7%) are observed when several complexes with the same aggregation state exist. A more accurate study of phenotypic plasticity shows that specific subunits can account for these differences (see below). Another possibility is the presence of adduct products such as small organic ions (lactate, urate or other) which are of functional and physiological relevance and which would be conserved in the physiological buffer but could be removed in the ESI-MS analysis conditions. In any case, these differences are small and the native state of observed products in ESI-MS is unambiguously verified by the agreement with MALLS determined masses. 6. USING MASS DATA TO EXPLORE DISSOCIA- TION/REASSOCIATION MECHANISMS IN GIANT HBL-HB 6.1. Interest of the Knowledge of Dissociation/ Reassociation Mechanisms Protein denaturing involves disruption of the higher order structure, such as their secondary and tertiary structure. It may be reversible or irreversible and be caused by temperature, pH, shear forces, surface area changes, surfactants, buffers, ionic strength, freeze-thaw cycles, organic solvents, and exposure to interfaces or denaturing chemicals often leading to aggregation and precipitation phenomena. Denaturation also typically involves reversible or irreversible unfolding depending on various factors. Size-exclusion chromatography (SEC) can provide information as to the levels of aggregation and fragmentation in a pharmaceutical protein, and combined with light scattering detection offers an easy, accurate and reliable alternative technique to investigate the association of macromolecules in solution. An additional benefit of this calculation lies in the molecular mass being simply that of the dissolved protein in solution. SEC- MALLS associated with ESI-MS analysis can provide useful results on the mechanism of dissociation/reassociation of HBL-Hb. We will illustrate these assumptions with the example of AmHb [6]. Polymerization is needed in extracellular respiratory proteins for retention in the vascular system and for adequate oxygen capacity at a manageable osmotic pressure, but this size requirement poses issues for a spontaneous assembly. The in vivo association of such complex proteins remains unclear in polychaete annelids. The pathway of folding of 87
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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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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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34 CHAPITRE 1. INTRODUCTION (a) (b)
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136 CHAPITRE 4. PLASTICITÉ PHÉNOT
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Dénaturant Natif
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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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