title: Surfactant protein C peptides with salt-bridges ("ion-locks") promote high surfactant activities by mimicking the α-helix and membrane topography of the native protein.
abstract: Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as 'helical adjuvants' to maintain activity by overriding the β-sheet propensities of the native sequences. Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges ("ion-locks") promote surface activity by mimicking the α-helix and membrane topography of native SP-C. Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu(-)-Lys(+) into the parent SP-C's. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency. Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with 'amyloid-like' properties. The enhanced β-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard (12)C-FTIR study showed that the α-helicity of these SP-C sequences in lipids was uniformly increased with Glu(-)-Lys(+) insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the α-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu(-)-Lys(+) ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C. Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest.
authors.affiliation: Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance, CA , United States of America ; Department of Pediatrics, David Geffen School of Medicine, University of California at Los Angeles , Los Angeles, CA , United States of America.
authors.forename: Frans J
authors.initials: FJ
authors.lastname: Walther
authors.affiliation: Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance, CA , United States of America ; Department of Pediatrics, David Geffen School of Medicine, University of California at Los Angeles , Los Angeles, CA , United States of America ; Department of Medicine, David Geffen School of Medicine, University of California at Los Angeles , Los Angeles, CA , United States of America ; Department of Physiology & Biophysics, School of Medicine, University of California , Irvine, CA , United States of America.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Waring
authors.affiliation: Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance, CA , United States of America.
authors.forename: José M
authors.initials: JM
authors.lastname: Hernández-Juviel
authors.affiliation: Department of Medicine, David Geffen School of Medicine, University of California at Los Angeles , Los Angeles, CA , United States of America.
authors.forename: Piotr
authors.initials: P
authors.lastname: Ruchala
authors.affiliation: Department of Pediatrics, University of Rochester , Rochester, NY , United States of America.
authors.forename: Zhengdong
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Pediatrics, University of Rochester , Rochester, NY , United States of America.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Notter
authors.affiliation: Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance, CA , United States of America.
authors.forename: Larry M
authors.initials: LM
authors.lastname: Gordon
keywords: Fourier Transform InfraRed spectroscopy; Membrane protein explorer program; PASTA algorithm; Protein synthesis; Salt-bridges; Surface activity; Surfactant protein C; Surfactant-deficient rabbit model; Synthetic surfactant
references.citation: Proc Natl Acad Sci U S A. 1987 Dec;84(24):8898-902
references.pmid: 3122208
references.citation: Proc Natl Acad Sci U S A. 1990 Apr;87(8):2985-9
references.pmid: 2326260
references.citation: Biochemistry. 2002 Aug 6;41(31):9852-62
references.pmid: 12146951
references.citation: Biophys J. 2000 Jan;78(1):458-65
references.pmid: 10620309
references.citation: J Biol Chem. 1996 Aug 9;271(32):19104-9
references.pmid: 8702584
references.citation: Neonatology. 2010 Jun;98(1):91-9
references.pmid: 20110733
references.citation: Biochim Biophys Acta. 1998 Nov 19;1408(2-3):79-89
references.pmid: 9813251
references.citation: Eur J Biochem. 2004 Jun;271(11):2086-92
references.pmid: 15153098
references.citation: Pept Res. 1991 Mar-Apr;4(2):95-101
references.pmid: 1815783
references.citation: Biochem Biophys Res Commun. 2010 May 21;396(1):2-6
references.pmid: 20494101
references.citation: Biopolymers. 1997 Aug;42(2):125-8
references.pmid: 9234991
references.citation: Biochemistry. 2007 Oct 2;46(39):11047-56
references.pmid: 17845058
references.citation: Am J Physiol Lung Cell Mol Physiol. 2010 Mar;298(3):L335-47
references.pmid: 20023175
references.citation: Biophys J. 2003 Oct;85(4):2417-29
references.pmid: 14507705
references.citation: Mol Genet Metab. 2000 Sep-Oct;71(1-2):342-51
references.pmid: 11001826
references.citation: Biophys J. 2002 Sep;83(3):1595-612
references.pmid: 12202384
references.citation: Biochemistry. 1994 May 17;33(19):6015-23
references.pmid: 8180229
references.citation: Biophys J. 1997 Jan;72(1):463-9
references.pmid: 8994633
references.citation: Exp Lung Res. 2005 Jul-Aug;31(6):563-79
references.pmid: 16019988
references.citation: Eur J Biochem. 2004 Jun;271(11):2076-85
references.pmid: 15153097
references.citation: Biochem J. 1995 Apr 15;307 ( Pt 2):535-41
references.pmid: 7733894
references.citation: Biophys J. 2010 Oct 6;99(7):2134-42
references.pmid: 20923647
references.citation: Crit Rev Biochem Mol Biol. 1995;30(2):95-120
references.pmid: 7656562
references.citation: Eur J Biochem. 1992 Jan 15;203(1-2):201-9
references.pmid: 1730226
references.citation: Biochim Biophys Acta. 1992 Apr 14;1138(4):261-7
references.pmid: 1562613
references.citation: J Biol Chem. 2003 Dec 26;278(52):52739-46
references.pmid: 14525980
references.citation: Biophys J. 2001 May;80(5):2262-72
references.pmid: 11325728
references.citation: Proc Natl Acad Sci U S A. 2012 Feb 14;109(7):2325-9
references.pmid: 22308375
references.citation: J Biol Chem. 2008 Apr 25;283(17):11126-34
references.pmid: 18303020
references.citation: Chem Phys Lipids. 1992 Nov;63(1-2):91-104
references.pmid: 1486663
references.citation: Biophys J. 2007 Jul 15;93(2):674-83
references.pmid: 17483162
references.citation: PLoS One. 2007 Oct 17;2(10):e1039
references.pmid: 17940603
references.citation: J Mol Biol. 1999 Feb 26;286(3):951-62
references.pmid: 10024461
references.citation: PLoS One. 2012;7(9):e44263
references.pmid: 22984481
references.citation: Biochemistry. 1991 Oct 15;30(41):10058-64
references.pmid: 1911771
references.citation: J Mol Biol. 2001 Oct 5;312(5):927-34
references.pmid: 11580239
references.citation: J Mol Biol. 1999 Nov 12;293(5):1241-55
references.pmid: 10547298
references.citation: Eur J Biochem. 1995 Apr 15;229(2):465-72
references.pmid: 7744069
references.citation: BMC Bioinformatics. 2007 Feb 27;8:65
references.pmid: 17324296
references.citation: Phys Rev E Stat Nonlin Soft Matter Phys. 2004 Feb;69(2 Pt 1):021602
references.pmid: 14995453
references.citation: Biochemistry. 2001 Oct 2;40(39):11876-86
references.pmid: 11570888
references.citation: Protein Sci. 2004 Dec;13(12):3314-21
references.pmid: 15537750
references.citation: Nat Rev Mol Cell Biol. 2007 Feb;8(2):101-12
references.pmid: 17245412
references.citation: Chem Phys Lipids. 1983 Jul;33(1):67-80
references.pmid: 6688762
references.citation: Eur J Biochem. 1998 Jul 1;255(1):116-24
references.pmid: 9692909
references.citation: Biochim Biophys Acta. 1998 Nov 19;1408(2-3):161-72
references.pmid: 9813304
references.citation: Protein Sci. 1997 Feb;6(2):416-21
references.pmid: 9041644
references.citation: J Pept Res. 2005 Dec;66(6):364-74
references.pmid: 16316452
references.citation: Pediatr Crit Care Med. 2013 Sep;14(7):657-65
references.pmid: 23846250
references.citation: Biochim Biophys Acta. 2012 Jan;1818(1):27-32
references.pmid: 21284935
references.citation: Nature. 2000 Jun 8;405(6787):647-55
references.pmid: 10864315
references.citation: Medchemcomm. 2011 Dec 1;2(12):1167-1173
references.pmid: 22530092
references.citation: Protein Sci. 1996 Aug;5(8):1662-75
references.pmid: 8844855
references.citation: Protein Sci. 2005 Oct;14(10):2582-9
references.pmid: 16155200
references.citation: Protein Sci. 2006 May;15(5):1127-32
references.pmid: 16597826
references.citation: Biochem J. 2009 Jul 15;421(3):415-23
references.pmid: 19435461
references.citation: PLoS One. 2010 Jan 13;5(1):e8672
references.pmid: 20084172
references.citation: Biochim Biophys Acta. 2012 Apr;1818(4):927-41
references.pmid: 22051023
references.citation: Biophys J. 2010 Nov 17;99(10):3234-43
references.pmid: 21081071
references.citation: FEBS Lett. 2002 Feb 13;512(1-3):47-51
references.pmid: 11852050
references.citation: Biochim Biophys Acta. 2008 Oct;1778(10):2127-37
references.pmid: 18515070
references.citation: Neonatology. 2007;91(4):303-10
references.pmid: 17575474
references.citation: Biochim Biophys Acta. 2010 Sep;1798(9):1632-50
references.pmid: 20435014
references.citation: FEBS Lett. 1999 Dec 31;464(3):138-42
references.pmid: 10618493
references.citation: Protein Eng. 2000 Mar;13(3):179-91
references.pmid: 10775659
references.citation: J Pept Res. 2000 Apr;55(4):330-47
references.pmid: 10798379
references.citation: Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7794-8
references.pmid: 7644495
references.citation: Eur J Biochem. 2002 Nov;269(22):5642-8
references.pmid: 12423364
references.citation: FEBS Lett. 1991 Apr 9;281(1-2):119-22
references.pmid: 2015882
references.citation: Biochemistry. 1996 Apr 23;35(16):5109-24
references.pmid: 8611495
references.citation: Am J Respir Crit Care Med. 2002 May 1;165(9):1201-2
references.pmid: 11991863
references.citation: Protein Eng Des Sel. 2007 Oct;20(10):521-3
references.pmid: 17720750
references.citation: Pediatrics. 2014 Jan;133(1):156-63
references.pmid: 24379227
references.citation: J Cell Biol. 2006 Jan 30;172(3):395-407
references.pmid: 16449190
references.citation: PLoS Comput Biol. 2006 Dec 15;2(12):e170
references.pmid: 17173479
references.citation: Proteins. 2006 Jun 1;63(4):1005-17
references.pmid: 16470585
references.citation: Biochemistry. 2008 Apr 8;47(14):4189-95
references.pmid: 18335996
references.citation: Eur Biophys J. 2007 Apr;36(4-5):477-89
references.pmid: 17051367
references.citation: Am J Respir Crit Care Med. 2011 Apr 15;183(8):966-8
references.pmid: 21498819
references.citation: Biophys J. 2001 Oct;81(4):2203-14
references.pmid: 11566791
references.citation: Biochem J. 2004 Jan 1;377(Pt 1):183-93
references.pmid: 14514353
references.citation: PeerJ. 2014 May 22;2:e393
references.pmid: 24883253
references.citation: Am J Respir Crit Care Med. 2011 Apr 15;183(8):1055-61
references.pmid: 21148720
references.citation: Biophys J. 2001 Jul;81(1):153-69
references.pmid: 11423403
references.citation: Biopolymers. 1986 Mar;25(3):469-87
references.pmid: 3697478
references.citation: Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2985-90
references.pmid: 8610155
references.citation: Neonatology. 2013;103(4):327-30
references.pmid: 23736010
references.citation: N Engl J Med. 2001 Feb 22;344(8):573-9
references.pmid: 11207353
references.citation: Protein Sci. 2009 Dec;18(12):2624-8
references.pmid: 19785006
references.citation: J Appl Physiol (1985). 2003 Nov;95(5):2055-63
references.pmid: 12897027
references.citation: Pediatr Res. 1998 Dec;44(6):860-4
references.pmid: 9853918