title: Probing the impact of GFP tagging on Robo1-heparin interaction.
abstract: Green fluorescent proteins (GFPs) and their derivatives are widely used as markers to visualize cells, protein localizations in in vitro and in vivo studies. The use of GFP fusion protein for visualization is generally thought to have negligible effects on cellular function. However, a number of reports suggest that the use of GFP may impact the biological activity of these proteins. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins mediating diverse patho-physiological processes. In the heparin-based interactome studies, heparin-binding proteins are often prepared as GFP fusion proteins. In this report, we use surface plasmon resonance (SPR) spectroscopy to study the impact of the GFP tagging on the binding interaction between heparin and a heparin-binding protein, the Roundabout homolog 1 (Robo1). SPR reveals that heparin binds with higher affinity to Robo1 than GFP-tagged Robo1 and through a different kinetic mechanism. A conformational change is observed in the heparin-Robo1 interaction, but not in the heparin-Robo1-GFP interaction. Furthermore the GFP-tagged Robo1 requires a shorter (hexasaccharide) than the tag-free Robo1 (octadecasaccharide). These data demonstrate that GFP tagging can reduce the binding affinity of Robo1 to heparin and hinder heparin binding-induced Robo1 conformation change.
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA, zhangf2@rpi.edu.
authors.forename: Fuming
authors.initials: F
authors.lastname: Zhang
authors.forename: Heather A
authors.initials: HA
authors.lastname: Moniz
authors.forename: Benjamin
authors.initials: B
authors.lastname: Walcott
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
authors.forename: Lianchun
authors.initials: L
authors.lastname: Wang
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000097905:Roundabout Proteins
chemical_list: D049452:Green Fluorescent Proteins; D006493:Heparin; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D000097905:Roundabout Proteins
references.citation: PLoS One. 2013;8(4):e50068
references.pmid: 23577054
references.citation: Proteins. 2005 Oct 1;61(1):115-26
references.pmid: 16080156
references.citation: PLoS One. 2009;4(2):e4433
references.pmid: 19209228
references.citation: Curr Opin Struct Biol. 2006 Dec;16(6):714-21
references.pmid: 17064887
references.citation: Biochem Biophys Res Commun. 1999 Jul 14;260(3):712-7
references.pmid: 10403831
references.citation: Mol Cell Proteomics. 2013 Aug;12(8):2160-73
references.pmid: 23649490
references.citation: Glycobiology. 1998 Dec;8(12):1183-94
references.pmid: 9858640
references.citation: Angew Chem Int Ed Engl. 2002 Feb 1;41(3):391-412
references.pmid: 12491369
references.citation: Anal Biochem. 2007 Mar 15;362(2):296-8
references.pmid: 17258166
references.citation: Bioinformatics. 2007 Sep 15;23(18):2376-84
references.pmid: 17709338
references.citation: Glycobiology. 2012 Sep;22(9):1183-92
references.pmid: 22641771
references.citation: Neurobiol Dis. 2004 Nov;17(2):310-8
references.pmid: 15474368
references.citation: Annu Rev Biomed Eng. 2006;8:181-231
references.pmid: 16834555
references.citation: Nat Biotechnol. 2006 Jan;24(1):79-88
references.pmid: 16369541
references.citation: Nat Methods. 2006 May;3(5):331
references.pmid: 16628201
references.citation: Science. 1996 Sep 6;273(5280):1392-5
references.pmid: 8703075
references.citation: J Cell Comp Physiol. 1962 Jun;59:223-39
references.pmid: 13911999
references.citation: J Cell Sci. 2010 Jul 1;123(Pt 13):2308-18
references.pmid: 20530570
references.citation: Nat Immunol. 2005 Sep;6(9):902-10
references.pmid: 16056228
references.citation: Cell. 1999 Mar 19;96(6):795-806
references.pmid: 10102268
references.citation: J Endocrinol. 2001 Aug;170(2):297-306
references.pmid: 11479127
references.citation: Glycobiology. 2004 Apr;14(4):17R-30R
references.pmid: 14718374
references.citation: Biochem Biophys Res Commun. 2000 Sep 16;276(1):292-7
references.pmid: 11006120
references.citation: J Biol Chem. 1992 Jun 25;267(18):12528-38
references.pmid: 1618758
references.citation: J Biol Chem. 2008 Jun 6;283(23):16226-34
references.pmid: 18359766
references.citation: Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):462-7
references.pmid: 11209050
references.citation: Nat Rev Immunol. 2006 Sep;6(9):633-43
references.pmid: 16917509
references.citation: PLoS One. 2006 Dec 20;1:e54
references.pmid: 17183684
references.citation: J Cell Sci. 2011 Jan 15;124(Pt 2):157-60
references.pmid: 21187342
references.citation: J Biol Chem. 2003 Nov 7;278(45):44626-31
references.pmid: 12909624
references.citation: Blood. 2009 Nov 5;114(19):4300-9
references.pmid: 19741192
references.citation: Nat Rev Mol Cell Biol. 2005 Jul;6(7):530-41
references.pmid: 16072037
references.citation: Nat Methods. 2009 Oct;6(10):713-17
references.pmid: 19953681
references.citation: Biochemistry. 2012 Jun 5;51(22):4618-26
references.pmid: 22574931
references.citation: Science. 1994 Feb 11;263(5148):802-5
references.pmid: 8303295
references.citation: J Virol. 2008 Sep;82(17):8911-6
references.pmid: 18524816
references.citation: Protein Sci. 1999 Apr;8(4):921-9
references.pmid: 10211839
references.citation: Nat Med. 2000 May;6(5):482-3
references.pmid: 10802676
references.citation: Biochimie. 2013 Dec;95(12):2345-53
references.pmid: 23994753
references.citation: J Biol Chem. 2005 Feb 18;280(7):5414-22
references.pmid: 15583000
references.citation: J Biol Chem. 2007 Apr 6;282(14):10465-71
references.pmid: 17289667
references.citation: Nat Med. 2008 Apr;14(4):448-53
references.pmid: 18345009