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The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 177 termining molecular masses, it can be used with great profit for unambiguous determination of subunit identity by their masses. In the previously mentioned study of Hc from a hydrothermal vent crab, subunit composition was also determined [74]. In both experienced conditions (hypoxia and hyperoxia), the same 4 polypeptide chains were detected. However, when comparing MaxEnt deconvoluted spectra from the two samples, a change in the abundance of one subunit can be observed: the 75 541 Da subunit is more abundant after hyperoxia. Since it has often been demonstrated that an oligo-hexameric state of arthropod Hc can depend on the presence or absence of certain subunit types, the observed change in the 75 541 Da subunit abundance could be suggested as a cause for the change in aggregation states within such a short period observed in the same experiment. Once again, even if interindividual variations could still have interfered with condition-induced variations, this results shows that a small change in subunit abundance can be detected by ESI-MS and encourage to perform such analysis on individual samples. This is made possible by the high sensitivity of the method and would permit to distinguish between interindividual variability and reproducible trends induced by experimental conditions. ESI-MS is a powerful method to explore subunit plasticity for these complexes, considering the similarities existing between subunits and the need to identify them precisely. CONCLUSION: WHICH QUESTIONS DO MALLS AND ESI-MS HELP TO SOLVE ? All the results presented here clearly reveal the complementarities of ESI-MS and MALLS analysis to investigate the structure of noncovalent multimeric complexes such as HBL-Hb and Hc. Indeed, the molecular masses of native proteins determined by light scattering systems agree well with known molecular masses based on gel permeation chromatography or analytical centrifugation, and LS technologies can be alternatives to these conventional methods. In theory, SEC- MALLS provides the capability of determining the “absolute” molecular masses of proteins and their protein-protein complexes as it is in solution. Molecular masses determined by SEC-MALLS depend only on the readings obtained from the downstream MALLS and refractive index (RI) detectors and not on the SEC elution position [38-41]. As for analytical centrifugation, the resulting average mass is independent of any external calibration. Other advantages of SEC-MALLS are that it is a “non-invasive” technique without incorporation of a radioactive or fluorescent tag and it is non-destructive. The sample may thus be recovered for use in subsequent studies. Compared with techniques such as analytical centrifugation, SEC-MALLS is more rapid and samples may be analyzed easily at various pH values, ionic strengths, and temperature and in the presence or absence of ligands. SEC-MALLS is a particularly useful tool for studying homoassociations and heteroassociations of proteins and other biological macromolecules [38-41, 137] as illustrated with AmHb dissociation/reassociation mechanism. In conclusion, SEC-MALLS offers a powerful tool for characterization of the biophysical properties of such proteins and their biologically relevant complexes in solution. MALLS and analytical centrifugation are the techniques the most likely to keep molecules in their native state, in particular for fragile assemblies such as annelid HBL-Hb in which the central piece may be lost by other techniques. Concerning protein conformation, the MALLS system can measure the rms gyration radius (Rg) of globular proteins but values are generally below the angular variation detection limit of 10 nm, making the determination unreliable. Conformational changes in the protein molecules during aggregation/denaturation can be predicted from these measurements. More accurate measurements of the conformation of these molecules would require the determination of additional parameters such as hydrodynamic radius (Rh) and are limited by difficulties in accurately measuring Rg of these globular proteins by MALLS. Even if MALLS gave plausible information on the mass of the native pigment in solution, it lacks the resolution capability to test for the existence of similar-sized isoforms. This can be achieved with a variety of mass spectrometric (MS) approaches. Indeed, MS can be used to determine molecular masses more accurately than by SEC-MALLS, but the ESI process may break fragile noncovalent interactions and the reliability of the method must be tested first. The good agreement between MALLS and ESI-MS results suggests that the interactions maintaining the complex are conserved during ionization. Thus the species observed by ESI- MS are likely to be representative of the species in solution and the high mass accuracy of the method can be used to distinguish between very close isoforms. The utility of MS for detection and quantification of noncovalent protein-protein interactions under native, equilibrium conditions in solution can be limited by several factors. Although the m/z range of the time-of-flight analyzer is theoretically unlimited, in practice it is often difficult to detect larger proteins and noncovalently bound macromolecular complexes in excess of 100kDa as their flight through technique used for improving the detection of these large structures is collisional cooling [138-143]. However, this seems to be protein dependent as revealed by the analysis of hemocyanin complexes presented in this review. In this case, ESI-MS in native mode enables to measure absolute mass of native complexes similar to the species in solution. MALLS has the advantage that it can be performed on-line with separative techniques such as FPLC using a physiological buffer as eluent, thus minimizing sample manipulation and possible deterioration, but does not permit to distinguish isoforms. When native masses can be determined by both techniques, small differences (2 to 4 % for Carcinus maenas) are often observed between MALLS and ESI-MS, with MALLS masses often being superior to MS masses. Two hypotheses can be proposed to explain this. First, as the two techniques are based on different physical principles, the differences could be inherent to the devices themselves and reflect no real difference. On the other hand, one can consider that during the ionization process in ESI-MS, labile solvent molecules and ions could be separated from the protein complex during vaporization in the nebulization chamber, while the MALLS measurement could take into account divalent cations or other physiological adducts and solvating layer 98
178 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al. involved in the pigment structure in the physiological buffer, hopefully the same as in the hemolymph. This is supported by the progressive decrease in mass observed with increasing denaturation/desolvatation: native MALLS mass is superior to native ESI-MS mass, which is superior to model mass from denatured subunits (Fig. 7). It is thus possible that what is really measured is not always exactly the same from one method to another. However, the good agreement between these two methods using different principles suggest that both of them provides accurate results and support further use of them in a complementary approach to study macromolecular complexes and protein-protein interactions. ACKNOWLEDGEMENTS The authors would like to thank their academic structures (CNRS, UPMC, ULP) for supporting their work. M.B. was funded by a MRT grant, n°18213-2005. ABBREVIATIONS Hb = Hemoglobin HBL-Hb = Hexagonal bilayer hemoglobin Chl = Chlorocruorin Hc = Hemocyanin TEM = Transmission electron microscopy STEM = Scanning transmission electron microscopy ESI-MS = Electrospray ionization mass spectrometry FFF = Field flow fractionation MALLS = Multi-angle laser light scattering n 50 = Hill-coefficient at half-saturation P 50 = Oxygen partial pressure at half-saturation LS = Light scattering HPLC = High performance liquid chromatography SDS-PAGE = Sodium dodecyl sulfate polyacrylamide gel electrophoresis rms = Root mean square Rg = Gyration radius Rh = Hydrodynamic radius cryo-EM = Cryoelectron microscopy AmHb = Arenicola marina hemoglobin LtHb = Lumbricus terrestris hemoglobin SAXS = Small-angle x-ray scattering. REFERENCES [1] Toulmond, A. and Truchot, J.P. (1993) La Recherche, 254, 562- 570. [2] Kurtz, D.M., Jr. (1992) In Blood and tissue oxygen carriers (Mangum, C.P., Ed.), pp. 151-171, Springer-Verlag, Berlin. [3] van Holde, K.E., Miller, K.I. and Decker, H. (2001) J. Biol. Chem., 276, 15563-15566. [4] Weber, R.E. and Vinogradov, S.N. (2001) Physiol. Rev., 81, 569- 628. [5] Katta, V. and Chait, B.T. (1991) J. Am. Chem. Soc., 113, 8534- 8535. [6] Rousselot, M., Le Guen, D. and Zal, F. (2006) FEBS J., 273, 1582- 1596. [7] Zal, F., Lallier, F.H. and Toulmond, A. (2000) International patent, PCT/FR01/01505 [8] Rousselot, M., Delpy, E., Drieu La Rochelle, C., Lagente, V., Pirow, R., Rees, J.F., Hagege, A., Le Guen, D., Hourdez, S. and Zal, F. (2006) Biotechnol. J., 1, 333-345. [9] Martin, A.G., Depoix, F., Stohr, M., Meissner, U., Hagner-Holler, S., Hammouti, K., Burmester, T., Heyd, J., Wriggers, W. and Markl, J. (2007) J. Mol. Biol., 366, 1332-1350. [10] Menze, M.A., Hellmann, N., Decker, H. and Grieshaber, M.K. (2005) Biochemistry, 44, 10328-10338. [11] van Holde, K.E., Miller, K.I. and van Olden, E. (2000) Biophys. Chem., 86, 165-172. [12] Decker, H. and Sterner, R. (1990) J. Mol. Biol., 211, 281-293. [13] Mangum, C.P. (1976) In Adaptation to environment: physiology of marine animals. (Newell, P.C., Ed.), pp. 191, Butterworth's: London, London. [14] Weber, R.E. (1978) in Physiology of annelids (Mill, P., Ed.), pp. 393-446, Academic Press, New York. [15] Svedberg, T. and Eriksson, I.B. (1933) J. Am. Chem. Soc., 55, 2834-2841. [16] Roche, J. (1965) in Studies in comparative biochemistry. (Munday, D.A., Ed.), Pergamon Press, Oxford. [17] Terwilliger, N.B. (1992) In Blood and tissue oxygen carriers (Mangum, C.P., Ed.), pp. 193-229, Springer-Verlag, Berlin. [18] Svedberg, T. and Eriksson, I.B. (1933) J. Am. Chem. Soc., 56, 1700-1705. [19] Mangum, C.P., Woodin, B.R., Bonaventura, C., Sullivan, B. and Bonaventura, J. (1975) Comp. Biochem. Physiol., 51A, 281-294. [20] Weber, R.E. and Baldwin, J. (1985) Mol. Physiol., 7, 93-106. [21] Antonini, E., Rossi-Fanelli, A. and Caputo, A. (1962) Arch. Biochem. Biophys., 97, 343-350. [22] Zhu, H., Ownby, D.W., Riggs, C.K., Nolasco, N.J., Stoops, J.K. and Riggs, A.F. (1996) J. Biol. Chem., 271, 30007-30021. [23] Martin, P.D., Kuchumov, A.R., Green, B.N., Oliver, R.W., Braswell, E.H., Wall, J.S. and Vinogradov, S.N. (1996) J. Mol. Biol., 255, 154-169. [24] Truchot, J.P. (1992) In Blood and tissue oxygen carriers. (Mangum, C.P., Ed.), pp. 377-410, Springer-Verlag, Berlin. [25] Bridges, C.R. (2001) J. Exp. Biol., 204, 1021-1032. [26] Mangum, C.P. and Rainer, J.S. (1988) Bio. Bull., 174, 77-82. [27] Bellelli, A., Giardina, B., Corda, M., Pellegrini, M.G., Cau, A., Condó, S.G. and Brunori, M. (1988) Comp. Biochem. Physiol., 91A, 445-449. [28] Condó, S.G., Pellegrini, M.G., Corda, M., Sanna, M.T., Cau, A. and Giardina, B. (1991) Biochem. J., 277, 419-421. [29] Mangum, C.P. (1994) Comp. Biochem. Physiol. Biochem. Mol. Biol., 108, 537-541. [30] Howlett, G.J., Minton, A.P. and Rivas, G. (2006) Curr. Opin. Chem. Biol., 10, 430-436. [31] Lebowitz, J., Lewis, M.S. and Schuck, P. (2002) Protein Sci., 11, 2067-2079. [32] Hanin, L., Green, B., Zal, F. and Vinogradov, S. (2003) J. Biosci., 28, 557-568. [33] Lee, S.C. and Whitaker, J.R. (2004) J. Agric. Food Chem., 52, 4948-4952. [34] Philo, J.S. (2006) AAPS J., 8, E564-571. [35] Müller, S.A. and Engel, A. (2001) Micron, 32, 21-31. [36] Schlags, W., Walther, M., Masree, M., Kratzel, M., Noe, C.R. and Lachmann, B. (2005) Electrophoresis, 26, 2461-2469. [37] Lamy, J.N., Green, B.N., Toulmond, A., Wall, J.S., Weber, R.E. and Vinogradov, S.N. (1996) Chem. Rev., 96, 3113-3124. [38] Harding, S.E. and Jumel, K. (1998) Curr. Protocols Protein Sci., 7.8.1-7.8.14. [39] Wyatt, P.J. (1993) Anal. Chim. Acta, 272, 1-40. [40] Takagi, T. (1990) J. Chromatogr., 506, 409-416. [41] Wen, J., Arakawa, T. and Philo, J.S. (1996) Anal. Biochem., 240, 155-166. [42] Doty, P.M., Zimm, B.H. and Mark, H. (1944) J. Chem. Phys., 12, 144-145. [43] Zimm, B.H. (1948) J. Chem. Phys., 16, 1093-1116. [44] Andersson, M., Wittgren, B. and Wahlund, K.G. (2003) Anal. Chem., 75, 4279-4291. [45] Silveira, J.R., Raymond, G.J., Hughson, A.G., Race, R.E., Sim, V.L., Hayes, S.F. and Caughey, B. (2005) Nature, 437, 257-261. 99
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THESE DE DOCTORAT DE L’UNIVERSITE
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i Résumé / Abstract Réponse adap
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(a) FIG. 1.6 - Fonctionnement d’u
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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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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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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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