Advertisement

Novel approaches to probe the binding of recoverin to membranes

  • Kim Potvin-Fournier
  • Geneviève Valois-Paillard
  • Marie-Claude Gagnon
  • Thierry Lefèvre
  • Pierre Audet
  • Line Cantin
  • Jean-François Paquin
  • Christian Salesse
  • Michèle Auger
Original Article
  • 83 Downloads

Abstract

Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. We have investigated the interaction of recoverin with zwitterionic phosphatidylcholine bilayers, the major lipid component of the rod outer segment disk membranes, using both 31P and 19F solid-state nuclear magnetic resonance (NMR) and infrared spectroscopy. In particular, several novel approaches have been used, such as the centerband-only detection of exchange (CODEX) technique to investigate lipid lateral diffusion and 19F NMR to probe the environment of the recoverin myristoyl group. The results reveal that the lipid bilayer organization is not disturbed by recoverin. Non-myristoylated recoverin induces a small increase in lipid hydration that appears to be correlated with an increased lipid lateral diffusion. The thermal stability of recoverin remains similar in the absence or presence of lipids and Ca2+. Fluorine atoms have been strategically introduced at positions 4 or 12 on the myristoyl moiety of recoverin to, respectively, probe its behavior in the interfacial and more hydrophobic regions of the membrane. 19F NMR results allow the observation of the calcium–myristoyl switch, the myristoyl group experiencing two different environments in the absence of Ca2+ and the immobilization of the recoverin myristoyl moiety in phosphatidylcholine membranes in the presence of Ca2+.

Keywords

Phosphatidylcholine Infrared spectroscopy Solid-state nuclear magnetic resonance spectroscopy Lateral diffusion of lipids Multilamellar vesicles Fluorine 

Abbreviations

19F

Fluorine-19

2H

Deuterium

31P

Phosphorus-31

Ca2+

Calcium ion

CODEX

Centerband-only detection of exchange

D2O

Deuterium oxide

DOPC

1,2-dioleoyl-sn-glycero-3-phosphatidylcholine

EGTA

Ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid

FT-IR

Fourier transform infrared

FWHM

Full width at half maximum

HEPES

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

HPLC

High-performance liquid chromatography

IR

Infrared

MAS

Magic-angle spinning

MLV

Multilamellar vesicle

NCS

Neuronal calcium sensor

NMR

Nuclear magnetic resonance

PC

Phosphatidylcholine

Rec

Recoverin

Rec-Myr

Myristoylated recoverin

Rec-Myr4F

Recoverin with a fluorine atom at position 4 in the myristoyl moiety

Rec-Myr12F

Recoverin with a fluorine atom at position 12 in the myristoyl moiety

Rec-nMyr

Non-myristoylated recoverin

ROS

Rod outer segment

Tm

Gel-to-fluid phase transition temperature

TPPM

Two-pulse phase-modulated

ν(C=O)

Carbonyl stretching vibration

νs(CH2)

CH2 symmetric stretching vibration

Notes

Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (J.-F.P., C.S. and M.A.), the Fonds de recherche du Québec-Nature et Technologies (FRQ-NT) (J.-F.P. and M.A.), and Université Laval (J.-F.P., C.S. and M.A.). The Regroupement québécois de recherche sur la fonction, l’ingénierie et les applications des protéines (PROTEO), the Centre de recherche sur les matériaux avancés (CERMA), the Centre québécois sur les matériaux fonctionnels (CQMF), and the Centre de recherche du CHU de Québec are acknowledged for the infrastructure provided to us. PROTEO and CQMF are supported by FRQ-NT. K.P.-F. is the recipient of graduate scholarships from NSERC and FRQ-NT. G.V.-P. is the recipient of a graduate scholarship from the Fonds de recherche du Québec-Santé (FRQS). M.-C.G. is the recipient of graduate scholarships from PROTEO and NSERC. The authors would like to thank Jean-François Rioux-Dubé and François Paquet-Mercier for their support with the FT-IR experiments, and Matthieu Fillion for helpful discussions.

Compliance with Ethical Standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

249_2018_1304_MOESM1_ESM.pdf (584 kb)
Supplementary material 1 (PDF 584 kb)

References

  1. Ames JB, Ikura M (2002) Structure and membrane-targeting mechanism of retinal Ca2+-binding proteins, recoverin and GCAP-2. Adv Exp Med Biol 514:333–348CrossRefPubMedGoogle Scholar
  2. Ames JB, Lim S (2012) Molecular structure and target recognition of neuronal calcium sensor proteins. Biochim Biophys Acta 1820:1205–1213CrossRefPubMedGoogle Scholar
  3. Ames JB, Porumb T, Tanaka T, Ikura M, Stryer L (1995) Amino-terminal myristoylation induces cooperative calcium-binding to recoverin. J Biol Chem 270:4526–4533CrossRefPubMedGoogle Scholar
  4. Ames JB, Ishima R, Tanaka T, Gordon JI, Stryer L, Ikura M (1997) Molecular mechanics of calcium-myristoyl switches. Nature 389:198–202CrossRefPubMedGoogle Scholar
  5. Arrondo JLR, Muga A, Castresana J, Goni FM (1993) Quantitative studies of the structure of proteins in solution by Fourier-transform infrared-spectroscopy. Prog Biophys Mol Biol 59:23–56CrossRefPubMedGoogle Scholar
  6. Bann JG, Pinkner J, Hultgren SJ, Frieden C (2002) Real-time and equilibrium 19F-NMR studies reveal the role of domain-domain interactions in the folding of the chaperone PapD. Proc Natl Acad Sci USA 99:709–714CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG (1995) Heteronuclear decoupling in rotating solids. J Chem Phys 103:6951–6958CrossRefGoogle Scholar
  8. Bentham M, Mazaleyrat S, Harris M (2006) Role of myristoylation and N-terminal basic residues in membrane association of the human immunodeficiency virus type 1 Nef protein. J Gen Virol 87:563–571CrossRefPubMedGoogle Scholar
  9. Blume A, Hübner W, Messner G (1988) Fourier transform infrared spectroscopy of 13C=O-labeled phospholipids hydrogen bonding to carbonyl groups. Biochemistry 27:8239–8249CrossRefPubMedGoogle Scholar
  10. Boman AL, Kahn RA (1995) ARF proteins—the membrane traffic police. Trends Biochem Sci 20:147–150CrossRefPubMedGoogle Scholar
  11. Burgoyne RD (2004) The neuronal calcium-sensor proteins. Biochim Biophys Acta 1742:59–68CrossRefGoogle Scholar
  12. Burgoyne RD, Haynes LP (2012) Understanding the physiological roles of the neuronal calcium sensor proteins. Mol Brain 5:11CrossRefGoogle Scholar
  13. Calvez P, Schmidt TF, Cantin L, Klinker K, Salesse C (2016) Phosphatidylserine allows observation of the calcium-myristoyl switch of recoverin and its preferential binding. J Am Chem Soc 138:13533–13540CrossRefGoogle Scholar
  14. Carafoli E, Krebs J (2016) Why calcium? How calcium became the best communicator. J Biol Chem 291:20849–20857CrossRefPubMedPubMedCentralGoogle Scholar
  15. Casal HL, Mantsch HH (1984) Polymorphic phase-behavior of phospholipid-membranes studied by infrared-spectroscopy. Biochim Biophys Acta 779:381–401CrossRefPubMedGoogle Scholar
  16. Chen CK, Inglese J, Lefkowitz RJ, Hurley JB (1995) Ca2+-dependent interaction of recoverin with rhodopsin kinase. J Biol Chem 270:18060–18066CrossRefPubMedGoogle Scholar
  17. Dalvit C, Knapp S (2017) 19F NMR isotropic chemical shift for efficient screening of fluorinated fragments which are racemates and/or display multiple conformers. Magn Reson Chem 55:1091–1095CrossRefPubMedGoogle Scholar
  18. Danielson MA, Falke JJ (1996) Use of 19F NMR to probe protein structure and conformational changes. Annu Rev Biophys Biomol Struct 25:163–195CrossRefPubMedPubMedCentralGoogle Scholar
  19. deAzevedo ER, Hu WG, Bonagamba TJ, Schmidt-Rohr K (1999) Centerband-only detection of exchange: Efficient analysis of dynamics in solids by NMR. J Am Chem Soc 121:8411–8412CrossRefGoogle Scholar
  20. Dedios AC, Pearson JG, Oldfield E (1993) Secondary and tertiary structural effects on protein NMR chemical-shifts—an abinitio approach. Science 260:1491–1496CrossRefGoogle Scholar
  21. Desmeules P, Penney S, Salesse C (2006) Single-step purification of myristoylated and nonmyristoylated recoverin and substrate dependence of myristoylation level. Anal Biochem 349:25–32CrossRefPubMedGoogle Scholar
  22. Desmeules P, Penney SE, Desbat B, Salesse C (2007) Determination of the contribution of the myristoyl group and hydrophobic amino acids of recoverin on its dynamics of binding to lipid monolayers. Biophys J 93:2069–2082CrossRefPubMedPubMedCentralGoogle Scholar
  23. Filippov A, Oradd G, Lindblom G (2009) Effect of NaCl and CaCl2 on the lateral diffusion of zwitterionic and anionic lipids in bilayers. Chem Phys Lipids 159:81–87CrossRefPubMedGoogle Scholar
  24. Flaherty KM, Zozulya S, Stryer L, McKay DB (1993) Three-dimensional structure of recoverin, a calcium sensor in vision. Cell 75:709–716CrossRefPubMedGoogle Scholar
  25. Fliesler SJ, Anderson RE (1983) Chemistry and metabolism of lipids in the vertebrate retina. Prog Lipid Res 22:79–131CrossRefPubMedGoogle Scholar
  26. Gagnon MC, Turgeon B, Savoie JD, Parent JF, Auger M, Paquin JF (2014) Evaluation of the effect of fluorination on the property of monofluorinated dimyristoylphosphatidylcholines. Org Biomol Chem 12:5126–5135CrossRefPubMedGoogle Scholar
  27. Gagnon MC, Strandberg E, Ulrich AS, Paquin JF, Auger M (2018) New insights into the influence of monofluorination on dimyristoylphosphatidylcholine membrane properties: a solid-state NMR study. Biochim Biophys Acta 1860:654–663CrossRefPubMedGoogle Scholar
  28. Gerig JT (1989) Fluorine nuclear magnetic resonance of fluorinated ligands. Methods Enzymol 177:3–23CrossRefPubMedGoogle Scholar
  29. Gerig JT (1994) Fluorine NMR of proteins. Prog Nucl Magn Reson Spectrosc 26:293–370CrossRefGoogle Scholar
  30. Gifford JL, Walsh MP, Vogel HJ (2007) Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem J 405:199–221CrossRefPubMedGoogle Scholar
  31. Goormaghtigh E, Cabiaux V, Ruysschaert J-M (1994) Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. In: Hilderson HJ, Ralston GB (eds) Physicochemical methods in the study of biomembranes. Springer, Boston, pp 405–450CrossRefGoogle Scholar
  32. Guimond-Tremblay J, Gagnon MC, Pineault-Maltais JA, Turcotte V, Auger M, Paquin JF (2012) Synthesis and properties of monofluorinated dimyristoylphosphatidylcholine derivatives: potential fluorinated probes for the study of membrane topology. Org Biomol Chem 10:1145–1148CrossRefPubMedGoogle Scholar
  33. Hubner W, Blume A (1998) Interactions at the lipid-water interface. Chem Phys Lipids 96:99–123CrossRefGoogle Scholar
  34. Jackson M, Mantsch HH (1995) The use and miuse of FTIR spectroscopy in the determination of protein-structure. Crit Rev Biochem Mol Biol 30:95–120CrossRefPubMedGoogle Scholar
  35. Jackson M, Haris PI, Chapman D (1991) Fourier-transform infrared spectroscopic studies of Ca2+-binding proteins. Biochemistry 30:9681–9686CrossRefPubMedGoogle Scholar
  36. Junker M, Creutz CE (1993) Endonexin (Annexin-IV)-mediated lateral segregation of phosphatidylglycerol in phosphatidylglycerol phosphatidylcholine membranes. Biochemistry 32:9968–9974CrossRefPubMedGoogle Scholar
  37. Kauppinen JK, Moffatt DJ, Mantsch HH, Cameron DG (1981) Fourier self-deconvolution—a method for resolving intrinsically overlapped bands. Appl Spectrosc 35:271–276CrossRefGoogle Scholar
  38. Kawamura S, Cox JA, Nef P (1994) Inhibition of rhodopsin phosphorylation by non-myristoylated recombinant recoverin. Biochem Biophys Res Commun 203:121–127CrossRefPubMedGoogle Scholar
  39. Kitevski-LeBlanc JL, Prosser RS (2012) Current applications of 19F NMR to studies of protein structure and dynamics. Prog Nucl Magn Reson Spectrosc 62:1–33CrossRefPubMedGoogle Scholar
  40. Klenchin VA, Calvert PD, Bownds MD (1995) Inhibition of rhodopsin kinase by recoverin—further evidence for a negative feedback-system in phototransduction. J Biol Chem 270:16147–16152CrossRefPubMedGoogle Scholar
  41. Kodati VR, Lafleur M (1993) Comparison between orientational orders in fluid lipid bilayers. Biophys J 64:163–170CrossRefPubMedPubMedCentralGoogle Scholar
  42. Kohler G, Hering U, Zschornig O, Arnold K (1997) Annexin V interaction with phosphatidylserine-containing vesicles at low and neutral pH. Biochemistry 36:8189–8194CrossRefPubMedGoogle Scholar
  43. Krimm S, Bandekar J (1986) Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv Protein Chem 38:181–364CrossRefPubMedGoogle Scholar
  44. Lai A, Saleem Q, Macdonald PM (2015) Centerband-only-detection-of-exchange 31P nuclear magnetic resonance and phospholipid lateral diffusion: theory, simulation and experiment. Phys Chem Chem Phys 17:25160–25171CrossRefPubMedGoogle Scholar
  45. Lange C, Koch KW (1997) Calcium-dependent binding of recoverin to membranes monitored by surface plasmon resonance spectroscopy in real time. Biochemistry 36:12019–12026CrossRefPubMedGoogle Scholar
  46. Lewis R, McElhaney RN (1998) The structure and organization of phospholipid bilayers as revealed by infrared spectroscopy. Chem Phys Lipids 96:9–21CrossRefGoogle Scholar
  47. Lewis RN, McElhaney RN, Pohle W, Mantsch HH (1994) Components of the carbonyl stretching band in the infrared spectra of hydrated 1,2-diacylglycerolipid bilayers: a reevaluation. Biophys J 67:2367–2375CrossRefPubMedPubMedCentralGoogle Scholar
  48. Liu JJ, Horst R, Katritch V, Stevens RC, Wuthrich K (2012) Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335:1106–1110CrossRefPubMedPubMedCentralGoogle Scholar
  49. Macdonald PM, Saleem Q, Lai A, Morales HH (2013) NMR methods for measuring lateral diffusion in membranes. Chem Phys Lipids 166:31–44CrossRefPubMedGoogle Scholar
  50. Marsh D (1990) CRC handbook of lipid bilayers. CRC Press, Boca RatonGoogle Scholar
  51. Martin BA, Oxhorn BC, Rossow CR, Perrino BA (2001) A cluster of basic amino acid residues in calcineurin B participates in the binding of calcineurin to phosphatidylserine vesicles. J Biochem 129:843–849CrossRefPubMedGoogle Scholar
  52. McLaughlin S, Aderem A (1995) The myristoyl-electrostatic switch—a modulator of reversible protein-membrane interactions. Trends Biochem Sci 20:272–276CrossRefPubMedGoogle Scholar
  53. Moskes C, Burghaus PA, Wernli B, Sauder U, Durrenberger M, Kappes B (2004) Export of Plasmodium falciparum calcium-dependent protein kinase 1 to the parasitophorous vacuole is dependent on three N-terminal membrane anchor motifs. Mol Microbiol 54:676–691CrossRefPubMedGoogle Scholar
  54. Nullmeier M, Koliwer-Brandl H, Kelm S, Zagel P, Koch KW, Brand I (2011) Impact of strong and weak lipid-protein interactions on the structure of a lipid bilayer on a gold electrode surface. ChemPhysChem 12:1066–1079CrossRefPubMedGoogle Scholar
  55. Oldfield E (1995) Chemical-shifts and 3-dimensional protein structures. J Biomol NMR 5:217–225CrossRefPubMedGoogle Scholar
  56. Ozawa T, Fukuda M, Nara M, Nakamura A, Komine Y, Kohama K, Umezawa Y (2000) How can Ca2+ selectively activate recoverin in the presence of Mg2+? Surface plasmon resonance and FT-IR spectroscopic studies. Biochemistry 39:14495–14503CrossRefPubMedGoogle Scholar
  57. Permyakov SE et al (2012) Oxidation mimicking substitution of conservative cysteine in recoverin suppresses its membrane association. Amino Acids 42:1435–1442CrossRefPubMedGoogle Scholar
  58. Potvin-Fournier K, Lefèvre T, Picard-Lafond A, Valois-Paillard G, Cantin L, Salesse C, Auger M (2014) The thermal stability of recoverin depends on calcium binding and its myristoyl moiety as revealed by infrared spectroscopy. Biochemistry 53:48–56CrossRefPubMedGoogle Scholar
  59. Potvin-Fournier K et al (2016) Discriminating lipid—from protein–calcium binding to understand the interaction between recoverin and phosphatidylglycerol model membranes. Biochemistry 55:3481–3491CrossRefPubMedGoogle Scholar
  60. Potvin-Fournier K, Valois-Paillard G, Lefèvre T, Cantin L, Salesse C, Auger M (2017) Membrane fluidity is a driving force for recoverin myristoyl immobilization in zwitterionic lipids. Biochem Biophys Res Commun 490:1268–1273CrossRefPubMedGoogle Scholar
  61. Rance M, Byrd RA (1983) Obtaining high-fidelity spin-1/2 powder spectra in anisotropic media—phase-cycled Hahn echo spectroscopy. J Magn Reson 52:221–240Google Scholar
  62. Ray S et al (1992) Cloning, expression, and crystallization of recoverin, a calcium sensor in vision. Proc Natl Acad Sci USA 89:5705–5709CrossRefPubMedPubMedCentralGoogle Scholar
  63. Resh MD (1999) Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta-Mol Cell Res 1451:1–16CrossRefGoogle Scholar
  64. Roy A, Dutta R, Kundu N, Banik D, Sarkar N (2016) A comparative study of the influence of sugars sucrose, trehalose, and maltose on the hydration and diffusion of DMPC lipid bilayer at complete hydration: investigation of structural and spectroscopic aspect of lipid-sugar interaction. Langmuir 32:5124–5134CrossRefPubMedGoogle Scholar
  65. Saleem Q, Lai A, Morales HH, Macdonald PM (2012) Lateral diffusion of bilayer lipids measured via 31P CODEX NMR. Chem Phys Lipids 165:721–730CrossRefPubMedGoogle Scholar
  66. Saurel O, Cezanne L, Milon A, Tocanne JF, Demange P (1998) Influence of annexin V on the structure and dynamics of phosphatidylcholine/phosphatidylserine bilayers: a fluorescence and NMR study. Biochemistry 37:1403–1410CrossRefPubMedGoogle Scholar
  67. Savitzky A, Golay MJE (1964) Smoothing and differentiation of data by simplified least squares procedures. Anal Chem 36:1627–1639CrossRefGoogle Scholar
  68. Seelig J (1978) 31P Nuclear magnetic-resonance and head group structure of phospholipids in membranes. Biochim Biophys Acta 515:105–140CrossRefPubMedGoogle Scholar
  69. Seifert MH, Ksiazek D, Azim MK, Smialowski P, Budisa N, Holak TA (2002) Slow exchange in the chromophore of a green fluorescent protein variant. J Am Chem Soc 124:7932–7942CrossRefPubMedGoogle Scholar
  70. Speyer JB, Sripada PK, Dasgupta SK, Shipley GG, Griffin RG (1987) Magnetic orientation of sphingomyelin lecithin bilayers. Biophys J 51:687–691CrossRefPubMedPubMedCentralGoogle Scholar
  71. Surewicz WK, Mantsch HH, Chapman D (1993) Determination of protein secondary structure by Fourier-transform infrared-spectroscopy—a critical-assessment. Biochemistry 32:389–394CrossRefPubMedGoogle Scholar
  72. Tanaka T, Ames JB, Harvey TS, Stryer L, Ikura M (1995) Sequestration of the membrane-targeting myristoyl group of recoverin in the calcium-free state. Nature 376:444–447CrossRefPubMedGoogle Scholar
  73. Tsui FC, Sundberg SA, Hubbell WL (1990) Distribution of charge on photoreceptor disk membranes and implications for charged lipid asymmetry. Biophys J 57:85–97CrossRefPubMedPubMedCentralGoogle Scholar
  74. Ulrich AS (2005) Solid state 19F NMR methods for studying biomembranes. Prog Nucl Magn Reson Spectrosc 46:1–21CrossRefGoogle Scholar
  75. Valentine KG, Mesleh MF, Opella SJ, Ikura M, Ames JB (2003) Structure, topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers. Biochemistry 42:6333–6340CrossRefPubMedGoogle Scholar
  76. Wachowicz M, Gill L, White JL (2009) Polyolefin blend miscibility: polarization transfer versus direct excitation exchange NMR. Macromolecules 42:553–555CrossRefGoogle Scholar
  77. Weiergraber OH, Senin II, Philippov PP, Granzin J, Koch KW (2003) Impact of N-terminal myristoylation on the Ca(2 +)-dependent conformational transition in recoverin. J Biol Chem 278:22972–22979CrossRefPubMedGoogle Scholar
  78. Yang ST, Lim SI, Kiessling V, Kwon I, Tamm LK (2016) Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein. Sci Rep 6:13CrossRefGoogle Scholar
  79. Ye LB, Van Eps N, Zimmer M, Ernst OP, Prosser RS (2016) Activation of the A2A adenosine G-protein-coupled receptor by conformational selection. Nature 533:265–268CrossRefPubMedGoogle Scholar
  80. Zhou WJ, Parent LJ, Wills JW, Resh MD (1994) Identification of a membrane-binding domain within the amino-terminal region of human-immunodeficiency-virus type-1 GAG protein which interacts with acidic phospholipids. J Virol 68:2556–2569PubMedPubMedCentralGoogle Scholar
  81. Zozulya S, Stryer L (1992) Calcium myristoyl protein switch. Proc Natl Acad Sci USA 89:11569–11573CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2018

Authors and Affiliations

  • Kim Potvin-Fournier
    • 1
    • 2
  • Geneviève Valois-Paillard
    • 1
    • 2
  • Marie-Claude Gagnon
    • 1
    • 3
  • Thierry Lefèvre
    • 1
  • Pierre Audet
    • 4
  • Line Cantin
    • 2
  • Jean-François Paquin
    • 3
  • Christian Salesse
    • 2
  • Michèle Auger
    • 1
  1. 1.Département de chimie, Regroupement québécois de recherche sur la fonction, l’ingénierie et les applications des protéines (PROTEO), Centre de recherche sur les matériaux avancés (CERMA), Centre québécois sur les matériaux fonctionnels (CQMF)Université Laval, Pavillon Alexandre-VachonQuébecCanada
  2. 2.CUO-recherche, Centre de recherche du CHU de Québec, Hôpital du Saint-Sacrement, Département d’ophtalmologie, Faculté de médecine, PROTEOUniversité LavalQuébecCanada
  3. 3.Département de chimie, PROTEO, Centre in Green Chemistry and Catalysis (CGCC)Université Laval, Pavillon Alexandre-VachonQuébecCanada
  4. 4.Département de chimieUniversité Laval, Pavillon Alexandre-VachonQuébecCanada

Personalised recommendations