title: Multiparametric high-resolution imaging of native proteins by force-distance curve-based AFM.
abstract: A current challenge in the life sciences is to understand how the properties of individual molecular machines adjust in order to meet the functional requirements of the cell. Recent developments in force-distance (FD) curve-based atomic force microscopy (FD-based AFM) enable researchers to combine sub-nanometer imaging with quantitative mapping of physical, chemical and biological properties. Here we present a protocol to apply FD-based AFM to the multiparametric imaging of native proteins under physiological conditions. We describe procedures for experimental FD-based AFM setup, high-resolution imaging of proteins in the native unperturbed state with simultaneous quantitative mapping of multiple parameters, and data interpretation and analysis. The protocol, which can be completed in 1-3 d, enables researchers to image proteins and protein complexes in the native unperturbed state and to simultaneously map their biophysical and biochemical properties at sub-nanometer resolution.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
authors.forename: Moritz
authors.initials: M
authors.lastname: Pfreundschuh
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Martinez-Martin
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
authors.forename: Estefania
authors.initials: E
authors.lastname: Mulvihill
authors.affiliation: Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.
authors.forename: Susanne
authors.initials: S
authors.lastname: Wegmann
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
authors.forename: Daniel J
authors.identifier: 0000000330750665
authors.initials: DJ
authors.lastname: Muller
mesh_terms: D018625:Microscopy, Atomic Force / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D011506:Proteins / Q000648:ultrastructure*; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D011506:Proteins
references.citation: Nat Cell Biol. 2001 Jun;3(6):607-10
references.pmid: 11389447
references.citation: Mol Cell Proteomics. 2010 Aug;9(8):1678-88
references.pmid: 20562411
references.citation: Biochemistry. 1997 Jan 21;36(3):461-8
references.pmid: 9012661
references.citation: J Mol Biol. 2010 Feb 26;396(3):610-6
references.pmid: 20036258
references.citation: Biophys J. 1997 Jan;72(1):445-8
references.pmid: 8994631
references.citation: Structure. 2012 Dec 5;20(12):2185-90
references.pmid: 23159125
references.citation: EMBO J. 2002 Jul 15;21(14):3598-607
references.pmid: 12110573
references.citation: Methods Enzymol. 1974;31:667-78
references.pmid: 4418026
references.citation: PLoS One. 2012;7(1):e30204
references.pmid: 22295076
references.citation: EMBO J. 2004 Aug 18;23(16):3206-15
references.pmid: 15297878
references.citation: Nat Struct Biol. 2000 Aug;7(8):644-7
references.pmid: 10932247
references.citation: J Mol Biol. 1999 Jan 29;285(4):1347-51
references.pmid: 9917378
references.citation: Micron. 2002;33(4):385-97
references.pmid: 11814877
references.citation: Biophys J. 1994 Dec;67(6):2454-9
references.pmid: 7696484
references.citation: Nat Protoc. 2007;2(9):2191-7
references.pmid: 17853875
references.citation: J Struct Biol. 1997 Jul;119(2):172-88
references.pmid: 9245758
references.citation: Nat Nanotechnol. 2010 Mar;5(3):208-12
references.pmid: 20154686
references.citation: Nat Chem Biol. 2009 Jun;5(6):383-90
references.pmid: 19448607
references.citation: Biophys J. 1992 Aug;63(2):578-82
references.pmid: 19431843
references.citation: J Mol Biol. 1995 Jun 2;249(2):239-43
references.pmid: 7783190
references.citation: J Biol Chem. 2010 Aug 27;285(35):27302-27313
references.pmid: 20566652
references.citation: Science. 1992 May 22;256(5060):1180-4
references.pmid: 1589799
references.citation: ACS Nano. 2012 Jul 24;6(7):5952-60
references.pmid: 22695112
references.citation: J Histochem Cytochem. 2000 May;48(5):719-24
references.pmid: 10769056
references.citation: Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):496-501
references.pmid: 9012812
references.citation: Nucleic Acids Res. 1997 Aug 1;25(15):3095-101
references.pmid: 9224610
references.citation: EMBO J. 2003 Oct 1;22(19):5220-9
references.pmid: 14517259
references.citation: Science. 2006 Oct 27;314(5799):601-2
references.pmid: 17068247
references.citation: J Mol Biol. 2003 Apr 11;327(5):925-30
references.pmid: 12662920
references.citation: Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20802-7
references.pmid: 22135457
references.citation: Science. 1999 Mar 12;283(5408):1727-30
references.pmid: 10073936
references.citation: Cold Spring Harb Perspect Med. 2012 Jul;2(7):a006247
references.pmid: 22762014
references.citation: Biophys J. 1995 Aug;69(2):708-15
references.pmid: 8527684
references.citation: Nat Protoc. 2010 Jun;5(6):975-85
references.pmid: 20448543
references.citation: J Mol Biol. 1999 Jan 8;285(1):33-9
references.pmid: 9878384
references.citation: Science. 1994 Oct 14;266(5183):257-9
references.pmid: 7939660
references.citation: ACS Nano. 2013 Feb 26;7(2):1054-63
references.pmid: 23347043
references.citation: Nano Lett. 2013;13(11):5585-93
references.pmid: 24079830
references.citation: J Mol Biol. 2005 Apr 1;347(3):597-606
references.pmid: 15755453
references.citation: Nature. 2000 May 25;405(6785):418-9
references.pmid: 10839529
references.citation: Biophys J. 2009 Dec 2;97(11):2985-92
references.pmid: 19948128
references.citation: Angew Chem Int Ed Engl. 2011 May 2;50(19):4410-3
references.pmid: 21472832
references.citation: Sci Rep. 2013;3:1063
references.pmid: 23323207
references.citation: Nat Methods. 2008 Jun;5(6):491-505
references.pmid: 18511917
references.citation: Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):836-8
references.pmid: 11607452
references.citation: Nat Nanotechnol. 2012 Apr 01;7(4):217-26
references.pmid: 22466857
references.citation: Curr Opin Struct Biol. 2004 Apr;14(2):250-9
references.pmid: 15093841
references.citation: Nat Commun. 2013;4:2926
references.pmid: 24336094
references.citation: J Mol Biol. 2009 Aug 21;391(3):586-98
references.pmid: 19527729
references.citation: Science. 1989 Mar 24;243(4898):1586-9
references.pmid: 2928794
references.citation: Chem Soc Rev. 2014 Feb 21;43(4):1189-200
references.pmid: 23970346
references.citation: Nat Nanotechnol. 2006 Oct;1(1):3-5
references.pmid: 18654126
references.citation: Curr Opin Struct Biol. 1997 Apr;7(2):279-84
references.pmid: 9094323
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8473-8
references.pmid: 12070341
references.citation: Biophys J. 2006 Oct 1;91(7):2532-42
references.pmid: 16798815
references.citation: J Cell Sci. 2005 Nov 15;118(Pt 22):5315-23
references.pmid: 16263758
references.citation: Langmuir. 2011 Feb 15;27(4):1308-13
references.pmid: 21090659
references.citation: Cell. 2013 Aug 15;154(4):723-6
references.pmid: 23953107
references.citation: Science. 2007 May 25;316(5828):1144-8
references.pmid: 17525328
references.citation: Biophys J. 1995 May;68(5):1681-6
references.pmid: 7612811
references.citation: Trends Biotechnol. 1999 Apr;17(4):143-50
references.pmid: 10203772
references.citation: J Bacteriol. 1996 Jun;178(11):3025-30
references.pmid: 8655475
references.citation: Biophys J. 1991 Dec;60(6):1438-44
references.pmid: 19431815
references.citation: J Mol Biol. 2012 Sep 14;422(2):300-9
references.pmid: 22613761
references.citation: Nat Struct Biol. 2000 Sep;7(9):715-8
references.pmid: 10966636
references.citation: Angew Chem Int Ed Engl. 2011 Dec 9;50(50):12103-8
references.pmid: 22006839
references.citation: Curr Opin Struct Biol. 2012 Apr;22(2):187-96
references.pmid: 22365575
references.citation: Ultramicroscopy. 2011 Dec;111(12):1659-69
references.pmid: 22094372
references.citation: Biophys J. 1994 Jun;66(6):2159-65
references.pmid: 8075349
references.citation: Cell Biol Int. 1997 Nov;21(11):697-706
references.pmid: 9768468
references.citation: Science. 2004 Aug 20;305(5687):1147-50
references.pmid: 15326353
references.citation: Curr Opin Struct Biol. 2006 Aug;16(4):489-95
references.pmid: 16797964
references.citation: Science. 1994 Sep 9;265(5178):1577-9
references.pmid: 8079171
references.citation: Science. 1995 Apr 7;268(5207):92-4
references.pmid: 7701347
references.citation: Biophys J. 2001 Mar;80(3):1359-71
references.pmid: 11222297
references.citation: ACS Nano. 2013 Mar 26;7(3):2642-50
references.pmid: 23442147
references.citation: Nanoscale Res Lett. 2011 Mar 30;6(1):270
references.pmid: 21711775
references.citation: J Struct Biol. 1997 Jul;119(2):232-7
references.pmid: 9245763
references.citation: Nat Nanotechnol. 2008 May;3(5):261-9
references.pmid: 18654521
references.citation: Biochim Biophys Acta. 2000 Aug 30;1460(1):27-38
references.pmid: 10984588
references.citation: Eur Biophys J. 2005 Feb;34(1):91-6
references.pmid: 15257425
references.citation: Proc Natl Acad Sci U S A. 2013 Jan 22;110(4):E313-21
references.pmid: 23269837
references.citation: Nat Methods. 2011 Feb;8(2):123-7
references.pmid: 21278722
references.citation: Langmuir. 2009 Jul 7;25(13):7471-7
references.pmid: 19292499
references.citation: Methods Mol Biol. 2005;299:35-51
references.pmid: 15980594
references.citation: Biophys J. 1999 Aug;77(2):1150-8
references.pmid: 10423460
references.citation: Phys Rev Lett. 2011 May 13;106(19):198101
references.pmid: 21668203
references.citation: Biophys J. 2002 Dec;83(6):3578-88
references.pmid: 12496125
references.citation: Science. 1991 Sep 20;253(5026):1405-8
references.pmid: 1910206
references.citation: Biophys J. 2011 Feb 16;100(4):1100-8
references.pmid: 21320456
references.citation: Nucleic Acids Res. 1997 Nov 1;25(21):4379-84
references.pmid: 9336471
references.citation: Biochemistry. 1995 Apr 11;34(14):4610-6
references.pmid: 7718563
references.citation: J Biol Chem. 1986 May 5;261(13):6084-9
references.pmid: 3084478
references.citation: J Mol Biol. 1998;283(1):83-94
references.pmid: 9761675
references.citation: Phys Rev B Condens Matter. 1992 May 15;45(19):11226-11232
references.pmid: 10001046
references.citation: Structure. 2011 Nov 9;19(11):1562-72
references.pmid: 22078556
references.citation: Nature. 2005 Sep 29;437(7059):656-63
references.pmid: 16193040
references.citation: Nat Nanotechnol. 2009 Aug;4(8):514-7
references.pmid: 19662014
references.citation: J Struct Biol. 2012 Apr;178(1):1-7
references.pmid: 22369932
references.citation: Phys Rev Lett. 1986 Mar 3;56(9):930-933
references.pmid: 10033323
references.citation: Nature. 2010 Nov 4;468(7320):72-6
references.pmid: 20935627
references.citation: Nat Methods. 2013 Sep;10(9):847-54
references.pmid: 23985731
references.citation: Science. 1994 Apr 15;264(5157):415-7
references.pmid: 8153628
references.citation: J Mol Biol. 1995 May 5;248(3):507-12
references.pmid: 7752220
references.citation: Nat Rev Mol Cell Biol. 2011 Mar;12(3):163-76
references.pmid: 21326200
references.citation: Science. 2011 Aug 5;333(6043):755-8
references.pmid: 21817054
references.citation: PLoS Biol. 2009 May;7(5):e1000103
references.pmid: 19419241
references.citation: Nat Struct Biol. 1999 Apr;6(4):346-9
references.pmid: 10201403
references.citation: Proc Natl Acad Sci U S A. 2013 Feb 19;110(8):2798-803
references.pmid: 23388629
references.citation: Science. 2000 Apr 7;288(5463):143-6
references.pmid: 10753119
references.citation: Biophys J. 2001 Jun;80(6):3009-18
references.pmid: 11371473
references.citation: Curr Biol. 2007 Oct 23;17(20):1765-70
references.pmid: 17919908
references.citation: J Struct Biol. 1998;121(3):356-61
references.pmid: 9705879
references.citation: J Mol Biol. 1999 Feb 5;285(5):1903-9
references.pmid: 9925773
references.citation: FEBS Lett. 2001 Aug 31;504(3):219-22
references.pmid: 11532457
references.citation: J Struct Biol. 1999 Dec 30;128(3):243-9
references.pmid: 10633063
references.citation: Science. 1999 Jul 2;285(5424):100-2
references.pmid: 10390357
references.citation: Nat Methods. 2006 May;3(5):347-55
references.pmid: 16628204
references.citation: J Microsc. 2002 Jul;207(Pt 1):43-51
references.pmid: 12135458
references.citation: Nature. 2013 Jun 13;498(7453):190-7
references.pmid: 23739333
references.citation: J Mol Biol. 2006 Mar 24;357(2):644-54
references.pmid: 16434052
references.citation: J Biol Chem. 2007 Mar 23;282(12):8895-904
references.pmid: 17227765