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Characterisation of the salmon cystic fibrosis transmembrane conductance regulator protein for structural studies

1 Faculty of Life Sciences, University of Manchester, Manchester, M13 9PL, U.K.;
2 Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149, Genova, Italy;
3 U.O.C di Genetica Molecolare, Istituto Giannina Gaslini, 16147, Genova, Italy

Special Issues: Structure and function of membrane proteins: channels and transporters

The cystic fibrosis transmembrane conductance regulator protein (CFTR) is a chloride channel highly expressed in the gills of Salmo salar, with a role in osmoregulation. It shares 60% identity with the human CFTR channel, mutations to which can cause the common genetic disorder cystic fibrosis CF. The expression and localisation of salmon CFTR have been investigated, but the isolated protein has not been extensively characterised. Here we present a protocol for the purification of recombinant salmon CFTR, along with biophysical and structural characterisation of the purified protein. Salmon CFTR was overexpressed in Saccharomyces cerevisiae, solubilised in the detergent LPG-14 and chromatographically purified by nickel-affinity and size-exclusion chromatography methods. Prior to size-exclusion chromatography samples of salmon CFTR had low purity, and contained large quantities of aggregated protein. Compared to size-exclusion chromatography profiles of other orthologues of CFTR, which had less evidence of aggregation, salmon CFTR appeared to have lower intrinsic stability than human and platypus CFTR. Nonetheless, repeated size-exclusion chromatography allowed monodisperse salmon CFTR to be isolated, and multi-angle light scattering was used to determine its oligomeric state. The monodispersity of the sample and its oligomeric state were confirmed using cryo-electron microscopy and small-angle X-ray scattering (SAXS). These data were also processed to calculate a low-resolution structure of the salmon CFTR, which showed similar architecture to other ATP-binding cassette proteins.
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1. Plog S, Mundhenk L, Bothe MK, et al. (2010) Tissue and cellular expression patterns of porcine CFTR: similarities to and differences from human CFTR. J Histochem Cytochem 58: 785-797.    

2. Crawford I, Maloney PC, Zeitlin PL, et al. (1991) Immunocytochemical localization of the cystic fibrosis gene product CFTR. P Natl Acad Sci USA 88: 9262-9266.    

3. Gadsby DC, Nairn AC (1999) Regulation of CFTR Cl- ion channels by phosphorylation and dephosphorylation. Adv Sec Messenger Phosphoprotein Res 33: 79-106.    

4. Gadsby DC, Nairn AC (1999) Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol Rev 79: S77-S107.

5. Kirk KL, Wang W (2011) A unified view of cystic fibrosis transmembrane conductance regulator (CFTR) gating: combining the allosterism of a ligand-gated channel with the enzymatic activity of an ATP-binding cassette (ABC) transporter. J Biol Chem 286: 12813-12819.    

6. Quinton PM, Reddy MM (2000) CFTR, a rectifying, non-rectifying anion channel? J Korean Med Sci 15 Suppl: S17-20.

7. Goss CH, Ratjen F (2013) Update in cystic fibrosis 2012. Am J Resp Crit Care 187: 915-919.    

8. Welsh MJ, Ramsey BW (1998) Research on cystic fibrosis: a journey from the Heart House. Am J Resp Crit Care 157: S148-154.    

9. Hiroi J, McCormick SD (2012) New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish. Resp Physiol Neurobi 184: 257-268.    

10. Christensen AK, Hiroi J, Schultz ET, et al. (2012) Branchial ionocyte organization and ion-transport protein expression in juvenile alewives acclimated to freshwater or seawater. J Exp Biol 215: 642-652.    

11. Chen JM, Cutler C, Jacques C, et al. (2001) A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models. Mol Biol Evol 18: 1771-1788.    

12. Kiilerich P, Kristiansen K, Madsen SS (2007) Cortisol regulation of ion transporter mRNA in Atlantic salmon gill and the effect of salinity on the signaling pathway. J Endocrinol 194: 417-427.    

13. Nilsen TO, Ebbesson LO, Madsen SS, et al. (2007) Differential expression of gill Na+, K+-ATPase alpha- and beta-subunits, Na+, K+, 2Cl- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar. J Exp Biol 210: 2885-2896.    

14. Mio K, Ogura T, Mio M, et al. (2008) Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain. J Biol Chem 283: 30300-30310.    

15. Rosenberg MF, O'Ryan LP, Hughes G, et al. (2011) The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate. J Biol Chem 286: 42647-42654.    

16. Zhang L, Aleksandrov LA, Riordan JR, et al. (2011) Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling. BBA-Biomembranes 1808: 399-404.    

17. Awayn NH, Rosenberg MF, Kamis AB, et al. (2005) Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Biochem Soc T 33: 996-999.    

18. Lewis HA, Buchanan SG, Burley SK, et al. (2004) Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator. EMBO J 23: 282-293.    

19. Thibodeau PH, Brautigam CA, Machius M, et al. (2005) Side chain and backbone contributions of Phe508 to CFTR folding. Nat Struct Mol Biol 12: 10-16.    

20. Galeno L, Galfre E, Moran O (2011) Small-angle X-ray scattering study of the ATP modulation of the structural features of the nucleotide binding domains of the CFTR in solution. Eur Biophys J 40: 811-824.    

21. Galfre E, Galeno L, Moran O (2012) A potentiator induces conformational changes on the recombinant CFTR nucleotide binding domains in solution. Cell Mol Life Sci 69: 3701-3713.    

22. Marasini C, Galeno L, Moran O (2013) A SAXS-based ensemble model of the native and phosphorylated regulatory domain of the CFTR. Cell Mol Life Sci 70: 923-933.    

23. Hudson RP, Chong PA, Protasevich, II, et al. (2012) Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 287: 28480-28494.    

24. Huang P, Liu Q, Scarborough GA (1998) Lysophosphatidylglycerol: a novel effective detergent for solubilizing and purifying the cystic fibrosis transmembrane conductance regulator. Anal biochem 259: 89-97.    

25. Wiener MC (2004) A pedestrian guide to membrane protein crystallization. Methods 34: 364-372.    

26. Carpenter EP, Beis K, Cameron AD, et al. (2008) Overcoming the challenges of membrane protein crystallography. Curr Opin Struc Biol 18: 581-586.    

27. Dobrovetsky E, Menendez J, Edwards AM, et al. (2007) A robust purification strategy to accelerate membrane proteomics. Methods 41: 381-387.    

28. Granseth E, Seppala S, Rapp M, et al. (2007) Membrane protein structural biology--how far can the bugs take us? Mol Membr Biol 24: 329-332.    

29. Lewinson O, Lee AT, Rees DC (2008) The funnel approach to the precrystallization production of membrane proteins. J Mol Biol 377: 62-73.    

30. Graeslund S (2008) Protein production and purification. Nat Meth 5: 135-146.    

31. Mancia F, Love J (2010) High-throughput expression and purification of membrane proteins. J Struct Biol 172: 85-93.    

32. Aller SG, Yu J, Ward A, et al. (2009) Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 323: 1718-1722.    

33. Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14: 673-681.    

34. Sonoda Y, Cameron A, Newstead S, et al. (2010) Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins. FEBS Lett 584: 2539-2547.    

35. Sonoda Y, Newstead S, Hu NJ, et al. (2011) Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures. Structure 19: 17-25.    

36. Drew D, Newstead S, Sonoda Y, et al. (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3: 784-798.    

37. Newstead S, Kim H, von Heijne G, et al. (2007) High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 104: 13936-13941.    

38. Clark KM, Fedoriw N, Robinson K, et al. Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography. Protein Expres Purif 71: 207-223.

39. Slotboom DJ, Duurkens RH, Olieman K, et al. (2008) Static light scattering to characterize membrane proteins in detergent solution. Methods 46: 73-82.    

40. Ouano AC, Kaye W (1974) Gel-permeation chromatography: X. Molecular weight detection by low-angle laser light scattering. J Polym Sci: Polym Chem Edit 12: 1151-1162.

41. Miller JL, Tate CG (2011) Engineering an ultra-thermostable beta(1)-adrenoceptor. J Mol Biol 413: 628-638.    

42. Shibata Y, White JF, Serrano-Vega MJ, et al. (2009) Thermostabilization of the neurotensin receptor NTS1. J Mol Biol 390: 262-277.    

43. Tate CG, Schertler GF (2009) Engineering G protein-coupled receptors to facilitate their structure determination. Curr Opin Struc Biol 19: 386-395.    

44. Warne T, Serrano-Vega MJ, Tate CG, et al. (2009) Development and crystallization of a minimal thermostabilised G protein-coupled receptor. Protein Expres Purif 65: 204-213.    

45. Aleksandrov AA, Kota P, Cui L, et al. (2012) Allosteric modulation balances thermodynamic stability and restores function of DeltaF508 CFTR. J Mol Biol 419: 41-60.    

46. Huang P, Stroffekova K, Cuppoletti J, et al. (1996) Functional expression of the cystic fibrosis transmembrane conductance regulator in yeast. Biochim Biophys Acta 1281: 80-90.    

47. Bear CE, Li CH, Kartner N, et al. (1992) Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell 68: 809-818.    

48. Kogan I, Ramjeesingh M, Li C, et al. (2002) Studies of the molecular basis for cystic fibrosis using purified reconstituted CFTR protein. Method Mol Med 70: 143-157.

49. Bai J, Swartz DJ, Protasevich, II, et al. (2011) A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris. PloS One 6: e22577.    

50. O'Ryan L, Rimington T, Cant N, et al. (2012) Expression and purification of the cystic fibrosis transmembrane conductance regulator protein in Saccharomyces cerevisiae. J Vis Exp e3860.

51. Pollock N, Cant N, Rimington T, et al. (2014) Purification of the cystic fibrosis transmembrane conductance regulator protein expressed in Saccharomyces cerevisiae. J Vis Exp e51447.

52. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Method 9: 671-675.    

53. Sievers F, Wilm A, Dineen D, et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7: 539.

54. Sievers F, Higgins DG (2014) Clustal Omega, accurate alignment of very large numbers of sequences. Methods in molecular biology 1079: 105-116.    

55. Dawson RJP, Locher KP (2007) Structure of the multidrug ABC transporter Sav1866 from Staphylococcus aureus in complex with AMP-PNP. FEBS Lett 581: 935-938.    

56. Hammersley A, Svensson S, Hanfland M, et al. (1996) Two-Dimensional Detector Software: From Real Detector to Idealised Image or Two-Theta Scan. High Pressure Res 14: 325-348.

57. Mateu L, Luzzati V, Vargas R, et al. (1990) Order-disorder phenomena in myelinated nerve sheaths. II. The structure of myelin in native and swollen rat sciatic nerves and in the course of myelinogenesis. J Mol Biol 215: 385-402.

58. Luzzati V, Tardieu A (1980) Recent developments in solution x-ray scattering. Annu Rev Biophys Bioeng 9: 1-29.    

59. Petoukhov M, Svergun D (2007) Analysis of X-ray and neutron scattering from biomacromolecular solutions. Curr Opin Struc Biol 17: 562-571.    

60. Guinier A, Fournet G (1955) Small angle scattering of x-rays. New York: Wiley.

61. Feigin L, Svergun D (1987) Structure analysis by small-angle x.ray and neutron scattering. New York, London: Plenum Press.

62. Svergun D (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 25: 495-503.    

63. Dawson RJ, Locher KP (2006) Structure of a bacterial multidrug ABC transporter. Nature 443: 180-185.    

64. Franke D, Svergun D (2009) DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J Appl Crystallogr 42: 342-346.    

65. Tian C, Vanoye CG, Kang C, et al. (2007) Preparation, functional characterization, and NMR studies of human KCNE1, a voltage-gated potassium channel accessory subunit associated with deafness and long QT syndrome. Biochemistry 46: 11459-11472.    

66. Oliver RC, Lipfert J, Fox DA, et al. (2013) Dependence of micelle size and shape on detergent alkyl chain length and head group. PloS One 8: e62488.    

67. Yang Z, Wang C, Zhou Q, et al. (2014) Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains. Protein Sci 23: 769-789.    

68. Gulati S, Jamshad M, Knowles TJ, et al. (2014) Detergent-free purification of ABC (ATP-binding-cassette) transporters. Biochem J 461: 269-278.    

69. Lyman CP (1968) Body temperature of exhausted salmon. Copeia 1968: 631-633.    

70. Behrisch HW (1969) Temperature and the regulation of enzyme activity in poikilotherms. Fructose diphosphatase from migrating salmon. Biochem J 115: 687-696.

71. Handeland SO, Berge Ö, Björnsson BT, et al. (2000) Seawater adaptation by out-of-season Atlantic salmon (Salmo salar L.) smolts at different temperatures. Aquaculture 181: 377-396.

72. Hsu HH, Lin LY, Tseng YC, et al. (2014) A new model for fish ion regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes). Cell Tissue Res 357: 225-243.    

73. Moorman BP, Inokuchi M, Yamaguchi Y, et al. (2014) The osmoregulatory effects of rearing Mozambique tilapia in a tidally changing salinity. Gen Comp Endocrinol [in press].

74. Sucre E, Bossus M, Bodinier C, et al. (2013) Osmoregulatory response to low salinities in the European sea bass embryos: a multi-site approach. J Comp Physiol B 183: 83-97.    

75. Guggino WB, Stanton BA (2006) New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 7: 426-436.    

76. Venerando A, Franchin C, Cant N, et al. (2013) Detection of phospho-sites generated by protein kinase CK2 in CFTR: mechanistic aspects of Thr1471 phosphorylation. PloS One 8: e74232.    

Copyright Info: © 2014, Naomi L. Pollock, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (http://creativecommons.org/licenses/by/4.0)

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