title: Self-aggregation of a recombinant form of the propeptide NH2-terminal of the precursor of pulmonary surfactant protein SP-B: a conformational study.
abstract: A recombinant form of the peptide N-terminally positioned from proSP-B (SP-BN) has been produced in Escherichia coli as fusion with the Maltose Binding Protein, separated from it by Factor Xa cleavage and purified thereafter. This protein module is thought to control assembly of mature SP-B, a protein essential for respiration, in pulmonary surfactant as it progress through the progressively acidified secretory pathway of pneumocytes. Self-aggregation studies of the recombinant propeptide have been carried out as the pH of the medium evolved from neutral to moderately acid, again to neutral and finally basic. The profile of aggregation versus subsequent changes in pH showed differences depending on the ionic strength of the medium, low or moderate, and the presence of additives such as L-arginine (a known aggregation suppressor) and Ficoll 70 (a macromolecular crowder). Circular dichroism studies of SP-BN samples along the aggregation process showed a decrease in alpha-helical content and a concomitant increase in beta-sheet. Intrinsic fluorescence emission of SP-BN was dominated by the emission of Trp residues in neutral medium, being its emission maximum shifted to red at low pH, suggesting that the protein undergoes a pH-dependent conformational change that increases the exposure of their Trp to the environment. A marked increase in the fluorescence emission of the extrinsic probe bis-ANS indicated the exposure of hydrophobic regions of SP-BN at pH 5. The fluorescence of bis-ANS decreased slightly at low ionic strength, but to a great extent at moderate ionic strength when the pH was reversed to neutrality, suggesting that self-aggregation properties of the SP-BN module could be tightly modulated by the conditions of pH and the ionic environment encountered by pulmonary surfactant during assembly and secretion.
authors.affiliation: Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, Ciudad Universitaria, 28040, Madrid, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Bañares-Hidalgo
authors.forename: A
authors.initials: A
authors.lastname: Bolaños-Gutiérrez
authors.forename: F
authors.initials: F
authors.lastname: Gil
authors.forename: E J
authors.initials: EJ
authors.lastname: Cabré
authors.forename: J
authors.initials: J
authors.lastname: Pérez-Gil
authors.forename: P
authors.initials: P
authors.lastname: Estrada
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D037701:Pulmonary Surfactant-Associated Protein B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011663:Pulmonary Surfactants / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011498:Protein Precursors; D037701:Pulmonary Surfactant-Associated Protein B; D011663:Pulmonary Surfactants; D011993:Recombinant Fusion Proteins
references.citation: Respir Res. 2005 Jul 22;6:80
references.pmid: 16042774
references.citation: Dev Biol (Basel). 2003;112:81-97
references.pmid: 12762507
references.citation: Microb Cell Fact. 2005 Apr 22;4(1):11
references.pmid: 15847694
references.citation: Biopolymers. 1991 Jan;31(1):119-28
references.pmid: 2025683
references.citation: Semin Cell Dev Biol. 2002 Aug;13(4):263-70
references.pmid: 12243725
references.citation: Eur J Biochem. 2004 Sep;271(17):3580-7
references.pmid: 15317594
references.citation: Annu Rev Physiol. 2001;63:555-78
references.pmid: 11181967
references.citation: Biochemistry. 2000 May 30;39(21):6529-37
references.pmid: 10828969
references.citation: Biochemistry. 2001 Feb 20;40(7):2113-28
references.pmid: 11329280
references.citation: Nature. 2002 Apr 4;416(6880):507-11
references.pmid: 11932737
references.citation: Biochemistry. 1991 Jul 16;30(28):6917-21
references.pmid: 1648964
references.citation: Biochemistry. 1994 Jun 21;33(24):7536-46
references.pmid: 8011619
references.citation: J Biochem. 2002 Oct;132(4):591-5
references.pmid: 12359074
references.citation: Biophys J. 2004 Sep;87(3):1631-9
references.pmid: 15345542
references.citation: J Biol Chem. 2005 Oct 14;280(41):34747-54
references.pmid: 16041062
references.citation: J Biol Chem. 1997 Apr 11;272(15):9640-7
references.pmid: 9092492
references.citation: Endocrinology. 2002 Apr;143(4):1302-9
references.pmid: 11897686
references.citation: Biochim Biophys Acta. 2008 Jul-Aug;1778(7-8):1676-95
references.pmid: 18515069
references.citation: J Immunol. 2006 Jan 1;176(1):416-25
references.pmid: 16365435
references.citation: Protein Expr Purif. 2003 Mar;28(1):1-8
references.pmid: 12651100
references.citation: J Biol Chem. 2001 Jan 5;276(1):715-21
references.pmid: 11024038
references.citation: Biochemistry. 1969 Oct;8(10):3902-8
references.pmid: 4310322
references.citation: Biochem J. 2003 Jan 1;369(Pt 1):179-84
references.pmid: 12356334
references.citation: Nature. 1989 Nov 16;342(6247):291-3
references.pmid: 2812028
references.citation: J Mol Biol. 2007 Feb 9;366(1):295-306
references.pmid: 17161425
references.citation: J Biol Chem. 2001 Aug 31;276(35):33027-35
references.pmid: 11402049
references.citation: FEBS Lett. 1998 Feb 13;423(1):1-4
references.pmid: 9506830
references.citation: Int Arch Allergy Appl Immunol. 1966;30(5):446-55
references.pmid: 4163860
references.citation: Chem Phys Lipids. 2006 Jun;141(1-2):105-18
references.pmid: 16600200
references.citation: Curr Opin Struct Biol. 2001 Feb;11(1):114-9
references.pmid: 11179900
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9831-6
references.pmid: 12900507
references.citation: J Biol Chem. 1998 Jun 12;273(24):15183-91
references.pmid: 9614132
references.citation: J Pharm Sci. 2004 Jun;93(6):1390-402
references.pmid: 15124199
references.citation: J Biol Chem. 2004 Feb 13;279(7):5346-52
references.pmid: 14634010
references.citation: Proc Natl Acad Sci U S A. 2001 Nov 6;98(23):13060-5
references.pmid: 11687604
references.citation: Biochim Biophys Acta. 1996 Jul 24;1312(3):177-85
references.pmid: 8703986
references.citation: Eur J Biochem. 2004 Aug;271(15):3242-7
references.pmid: 15265043
references.citation: EMBO J. 2000 Aug 1;19(15):3870-5
references.pmid: 10921869
references.citation: Biophys J. 1985 Jun;47(6):765-72
references.pmid: 4016197
references.citation: Anal Biochem. 2000 Dec 15;287(2):252-60
references.pmid: 11112271
references.citation: Arch Biochem Biophys. 1997 May 1;341(1):17-24
references.pmid: 9143348
references.citation: Protein Sci. 2002 Aug;11(8):2022-32
references.pmid: 12142456
references.citation: J Biol Chem. 1996 Aug 16;271(33):19689-95
references.pmid: 8702672
references.citation: Am J Respir Cell Mol Biol. 2004 Apr;30(4):449-58
references.pmid: 12972403
references.citation: J Virol. 1999 Jun;73(6):4567-74
references.pmid: 10233915