European Biophysics Journal

, Volume 47, Issue 2, pp 165–177 | Cite as

Conformational changes, from β-strand to α-helix, of the fatty acid-binding protein ReP1-NCXSQ in anionic lipid membranes: dependence with the vesicle curvature

  • Vanesa V. Galassi
  • Silvina R. Salinas
  • Guillermo G. Montich
Original Article


We studied the conformational changes of the fatty acid-binding protein ReP1-NCXSQ in the interface of anionic lipid membranes. ReP1-NCXSQ is an acidic protein that regulates the activity of the Na+/Ca2+ exchanger in squid axon. The structure is a flattened barrel composed of two orthogonal β-sheets delimiting an inner cavity and a domain of two α-helix segments arranged as a hairpin. FTIR and CD spectroscopy showed that the interactions with several anionic lipids in the form of small unilamellar vesicles (SUVs) induced an increase in the proportion of helix secondary structure. Lower amount or no increase in α-helix was observed upon the interaction with anionic lipids in the form of large unilamellar vesicles (LUVs). The exception was 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) that was equally efficien to to induce the conformational change both in SUVs and in LUVs. In solution, the infrared spectra of ReP1-NCXSQ at temperatures above the unfolding displayed a band at 1617 cm−1 characteristic of aggregated strands. This band was not observed when the protein interacted with DMPG, indicating inhibition of aggregation in the interface. Similarly to the observed in L-BABP, another member of the fatty acid binding proteins, a conformational change in ReP1-NCXSQ was coupled to the gel to liquid-crystalline lipid phase transition.


ReP1-NCXSQ Lipid membrane Protein conformational change Infrared spectroscopy Circular dichroism Membrane curvature 



Regulatory protein of the squid nerve sodium calcium exchanger


Fatty acid-binding protein


Chicken liver bile acid-binding protein












1,2-dipalmitoyl -sn-glycero-3-phosphate






Large unilamellar vesicle


Fourier transform infrared


Circular dichroism


Fourier self-deconvolution



We thank Dr. G. Berberián and Dr. L. Beaugé for the kind donation of cDNA for ReP1-NCXSQ. This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Agencia Nacional de Promoción Científica y Tecnológica, AMPCyT, and Secretaría de Ciencia y Técnica-UNC, SECyT-UNC. CONICET also granted fellowships for VVG and SRS.


  1. Arrondo JL, Goñi FM (1999) Structure and dynamics of membrane proteins as studied by infrared spectroscopy. Prog Biophys Mol Biol 72:367–405CrossRefPubMedGoogle Scholar
  2. Barroso RP, Perez KR, Cuccovia IM, Lamy MT (2012) Aqueous dispersions of DMPG in low salt contain leaky vesicles. Chem Phys Lipids 65:169–177CrossRefGoogle Scholar
  3. Beaugé L, Dipolo R, Bollo M, Cousido A, Berberián G, Podjarny A (2013) Metabolic regulation of the squid nerve Na(+)/Ca (2+) exchanger: recent developments. Adv Exp Med Biol 961:149–161CrossRefPubMedGoogle Scholar
  4. Berberián G, Bollo M, Montich G, Roberts G, Degiorgis JA, DiPolo R, Beaugé L (2009) A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger. Biochim Biophys Acta 1788:1255–1262CrossRefPubMedGoogle Scholar
  5. Berberián G, Podjarny A, DiPolo R, Beaugé L (2012) Metabolic regulation of the squid nerve Na+/Ca2+ exchanger: recent kinetic, biochemical and structural developments. Prog Biophys Mol Biol 108:47–63CrossRefPubMedGoogle Scholar
  6. Berg OG, Gelb MH, Tsai MD, Jain MK (2001) Interfacial enzymology: the secreted phospholipase A2-paradigm. Chem Rev 9:2613–2654CrossRefGoogle Scholar
  7. Byler DM, Susi H (1986) Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers 25:469–487CrossRefPubMedGoogle Scholar
  8. Casal HL, Mantsch HH (1984) Polymorphic phase behaviour of phospholipid membranes studied by infrared spectroscopy. Biochim Biophys Acta 779:381–401CrossRefPubMedGoogle Scholar
  9. Chong SSY, Taneva SG, Lee JMC, Cornell RB (2014) The curvature sensitivity of a membrane-binding amphipathic helix can be modulated by the charge on a flanking region. Biochemistry 53:450–461CrossRefPubMedGoogle Scholar
  10. Cornell RB, Ridgway ND (2015) CTP:phosphocholine cytidylyltransferase: function, regulation, and structure of an amphitropic enzyme required for membrane biogenesis. Prog Lipid Res 59:147–171CrossRefPubMedGoogle Scholar
  11. Cousido-Siah A, Ayoub D, Berberián G, Bollo M, Van Dorsselaer A, Debaene F, DiPolo R, Petrova T, Schulze-Briese C, Olieric V, Esteves A, Mitschler A, Sanglier-Cianférani S, Beaugé L, Podjarny A (2012) Structural and functional studies of ReP1-NCXSQ, a protein regulating the squid nerve Na+/Ca2+ exchanger. Acta Crystallogr D Biol Crystallogr 68:1098–1107CrossRefPubMedGoogle Scholar
  12. De Gerónimo E, Hagan RM, Wilton DC, Córsico B (2010) Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes. Biochim Biophys Acta 1801:1082–1089CrossRefPubMedGoogle Scholar
  13. Decca MB, Perduca M, Monaco HL, Montich GG (2007) Conformational changes of chicken liver bile acid-binding protein bound to anionic lipid membrane are coupled to the lipid phase transitions. Biochim Biophys Acta 1768:1583–1591CrossRefPubMedGoogle Scholar
  14. Decca MB, Galassi VV, Perduca M, Monaco LH, Montich GG (2010) Influence of the lipid phase state and electrostatic surface potential on the conformations of a peripherally bound membrane protein. J Phys Chem B 46:15141–15150CrossRefGoogle Scholar
  15. Drake AF, Hider RC (1979) The structure of melittin in lipid bilayer membranes. Biochim Biophys Acta 555:371–373CrossRefPubMedGoogle Scholar
  16. Dyszy F, Pinto AP, Araújo AP, Costa-Filho AJ (2013) Probing the interaction of brain fatty acid binding protein (B-FABP) with model membranes. PLoS One 8:1–11CrossRefGoogle Scholar
  17. Enoki TA, Henriques VB, Lamy MT (2012) Light scattering on the structural characterization of DMPG vesicles along the bilayer anomalous phase transition. Chem Phys Lipids 165:826–837CrossRefPubMedGoogle Scholar
  18. Fanani ML, Hartel S, Maggio B, De Tullio L, Jara J, Olmos F, Oliveira RG (2010) The action of sphingomyelinase in lipid monolayers as revealed by microscopic image analysis. Biochim Biophys Acta 1798:1309–1323CrossRefPubMedGoogle Scholar
  19. Fidelio GD, Maggio B, Cumar FA (1982) Interaction of soluble and membrane proteins with monolayers of glycosphingolipids. Biochem J 203:717–725CrossRefPubMedPubMedCentralGoogle Scholar
  20. Galassi VV, Villarreal MA, Posada V, Montich GG (2014) Interactions of the fatty acid-binding protein ReP1-NCXSQ with lipid membranes. Influence of the membrane electric field on binding and orientation. Biochim Biophys Acta 1838:910–920CrossRefPubMedGoogle Scholar
  21. Gorbenko G, Trusova V (2011) Protein aggregation in a membrane environment. Adv Protein Chem Struct Biol 84:113–142CrossRefPubMedGoogle Scholar
  22. Johnson WC (1999) Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins: Struct Func Genet 35:307–312CrossRefGoogle Scholar
  23. Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22:2577–2637CrossRefPubMedGoogle Scholar
  24. Klocek G, Schulthess T, Shai Y, Seelig J (2009) Thermodynamics of melittin binding to lipid bilayers. Aggregation and pore formation. Biochemistry 48:2586–2596CrossRefPubMedGoogle Scholar
  25. Ladokhin AS, White SH (1999) Folding of amphipathic alpha-helices on membranes: energetics of helix formation by melittin. J Mol Biol 285:1363–1369CrossRefPubMedGoogle Scholar
  26. Lefèvre T, Subirade M (2001) Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane. Biochim Biophys Acta 1549:37–50CrossRefPubMedGoogle Scholar
  27. Loew C, Riske KA, Lamy MT, Seelig J (2011) Thermal phase behavior of DMPG bilayers in aqueous dispersions as revealed by 2H- and 31P-NMR. Langmuir 27:10041–10049CrossRefPubMedGoogle Scholar
  28. Meier M, Seelig J (2007) Thermodynamics of the coil <==> beta-sheet transition in a membrane environment. J Mol Biol 369:277–289CrossRefPubMedGoogle Scholar
  29. Micheletto MC, Mendes LFS, Basso LGM, Fonseca-Maldonado RG, Costa-Filho AJ (2017) Lipid membranes and acyl-CoA esters promote opposing effects on acyl-CoA binding protein structure and stability. Int J Biol Macromol 102:284–296CrossRefPubMedGoogle Scholar
  30. Nolan V, Perduca M, Monaco HL, Maggio B, Montich GG (2003) Interactions of chicken liver basic fatty acid-binding protein with lipid membranes. Biochim Biophys Acta 1611:98–106CrossRefPubMedGoogle Scholar
  31. Nuscher B, Kamp F, Mehnert T, Odoy S, Haass C, Kahle PJ, Beyer K (2004) Alpha-synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study. J Biol Chem 279:21966–21975CrossRefPubMedGoogle Scholar
  32. Paolorossi M, Montich GG (2011) Conformational changes of β2-human glycoprotein I and lipid order in lipid-protein complexes. Biochim Biophys Acta 1808:2167–2177CrossRefPubMedGoogle Scholar
  33. Perrin RJ, Woods WS, Clayton DF, George JM (2000) Interaction of human alpha-Synuclein and Parkinson’s disease variants with phospholipids. Structural analysis using site-directed mutagenesis. J Biol Chem 275:34393–34398CrossRefPubMedGoogle Scholar
  34. Provencher SW, Glöckner J (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20:33–37CrossRefPubMedGoogle Scholar
  35. Raimunda D, Bollo M, Beaugé L, Berberián G (2009) Squid nerve Na+/Ca2+ exchanger expressed in Saccharomyces cerevisiae: up-regulation by a phosphorylated cytosolic protein (ReP1-NCXSQ) is identical to that of native exchanger in situ. Cell Calcium 45:499–508CrossRefPubMedGoogle Scholar
  36. Ramirez F, Jain MK (1991) Phospholipase A2 at the bilayer interface. Proteins 4:229–239CrossRefGoogle Scholar
  37. Rey-Burusco MF, Ibáñez-Shimabukuro M, Gabrielsen M, Franchini GR, Roe AJ, Griffiths K, Zhan B, Cooper A, Kennedy MW, Córsico B, Smith BO (2015) Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus. Biochem J 471:403–414CrossRefPubMedPubMedCentralGoogle Scholar
  38. Riske KA, Amaral LQ, Dobereiner HG, Lamy MT (2004) Mesoscopic structure in the chain-melting regime of anionic phospholipid vesicles: DMPG. Biophys J 86:3722–3733CrossRefPubMedPubMedCentralGoogle Scholar
  39. Riske KA, Amaral LQ, Lamy MT (2009) Extensive bilayer perforation coupled with the phase transition region of an anionic phospholipid. Langmuir 25:10083–10091CrossRefPubMedGoogle Scholar
  40. Schneider MF, Marsh D, Jahn W, Kloesgen B, Heimburg T (1999) Network formation of lipid membranes: triggering structural transitions by chain melting. Proc Natl Acad Sci USA 25:14312–14317CrossRefGoogle Scholar
  41. Sreerama N, Woody RW (1993) A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal Biochem 209:32–44CrossRefPubMedGoogle Scholar
  42. Sreerama N, Venyaminov SY, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal Biochem 287:243–251CrossRefPubMedGoogle Scholar
  43. Storch J, McDermott L (2009) Structural and functional analysis of fatty acid-binding proteins. J Lipid Res 50(Suppl):S126–S131CrossRefPubMedPubMedCentralGoogle Scholar
  44. Surewicz WK, Leddy JJ, Mantsch HH (1990) Structure, stability, and receptor interaction of cholera toxin as studied by Fourier-transform infrared spectroscopy. Biochemistry 29:8106–8111CrossRefPubMedGoogle Scholar
  45. Touw WG, Baakman C, Black J, te Beek TAH, Krieger E, Robbie P, Vriend JG (2015) A series of PDB related databases for everyday needs. Nucleic Acids Research 43:D364–D368 (database issue) CrossRefPubMedGoogle Scholar
  46. Verger R (1997) ‘Interfacial activation’ of lipases: facts and artifacts. Trends Biotechnol 15:32–38CrossRefGoogle Scholar
  47. Villarreal MA, Perduca M, Monaco HL, Montich GG (2008) Binding and interactions of L-BABP to lipid membranes studied by molecular dynamic simulations. Biochim Biophys Acta 1778:1390–1397CrossRefPubMedGoogle Scholar
  48. Wang S-X, Sun Y-T, Sui S-F (2000) Membrane-induced conformational change in human apolipoprotein H. Biochem J 348:103–106CrossRefPubMedPubMedCentralGoogle Scholar
  49. Wimley WC, White SH (2004) Reversible unfolding of beta-sheets in membranes: a calorimetric study. J Mol Biol 342:703–711CrossRefPubMedPubMedCentralGoogle Scholar
  50. Wimley WC, Hristova K, Ladokhin AS, Silvestro L, Axelsen PH, White SH (1998) Folding of beta-sheet membrane proteins: a hydrophobic hexapeptide model. J Mol Biol 277:1091–1110CrossRefPubMedGoogle Scholar
  51. Wu JR, Lentz BR (1991) Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1. Biophys J 60:70–80CrossRefPubMedPubMedCentralGoogle Scholar
  52. Zhang X, Keiderling TA (2006) Lipid-induced conformational transitions of beta-lactoglobulin. Biochemistry 45:8444–8452CrossRefPubMedGoogle Scholar
  53. Zhang X, Ge N, Keiderling TA (2007) Electrostatic and hydrophobic interactions governing the interaction and binding of beta-lactoglobulin to membranes. Biochemistry 46:5252–5260CrossRefPubMedGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2017

Authors and Affiliations

  • Vanesa V. Galassi
    • 1
    • 2
    • 3
  • Silvina R. Salinas
    • 1
    • 2
    • 4
  • Guillermo G. Montich
    • 1
    • 2
  1. 1.Departamento de Química Biológica “Ranwel Caputto”, Facultad de Ciencias QuímicasUniversidad Nacional de CórdobaCórdobaArgentina
  2. 2.Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC)CONICET, Universidad Nacional de CórdobaCórdobaArgentina
  3. 3.CONICET, Facultad de Ciencias Exactas y NaturalesUniversidad Nacional de CuyoMendoza Argentina
  4. 4.Centro de Excelencia en Productos y Procesos Córdoba-CONICET, Pabellón CEPROCOR, X5164CórdobaArgentina

Personalised recommendations