Abstract
Atomic force microscopy (AFM) is one of the most versatile tools currently used in nanoscience. AFM allows for performing nondestructive imaging of almost any sample in either air or liquid, regardless whether the specimen is insulating, conductive, transparent, or opaque. It also allows for measuring interaction forces between a sharp probe and a sample surface, therefore allowing to probe nanomechanical properties of the specimen by either applying a controlled force or pulling the sample. It can provide topography, mechanical, magnetic, and conductive maps for very different type of samples. Transferred to the field of biology, today, AFM is the only microscopy technique able to produce images from biomolecules to bacteria and cells with nanometric resolution in aqueous media. Here, we will focus on the biological applications of AFM to flavoproteins. Despite references in the literature are scarce in this particular field, here it is described how imaging with AFM can contribute to describe catalysis mechanisms of some flavoenzymes, how oxidation states or binding of relevant ligands influence the association state of molecules, the dynamics of functional quaternary assemblies, and even visualize structural differences of individual protein molecules. Furthermore, we will show how force spectroscopy can be used to obtain the kinetic parameters, the dissociation landscape and the mechanical forces that maintain flavoprotein complexes, including the possibility to specifically detect particular flavoproteins on a sample.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Miller H, Zhou Z, Shepherd J, Wollman AJM, Leake MC (2017) Single-molecule techniques in biophysics: a review of the progress in methods and applications. Rep Prog Phys 81:24601
Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5:491–505
Binnig G, Quate CF, Gerber CH (1986) Atomic force microscope. Phys Rev Lett 56:930
Florin EL, Moy VT, Gaub HE (1994) Adhesion forces between individual ligand-receptor pairs. Science 264:415–417
Merkel R, Nassoy P, Leung L, Ritchie K, Evans E (1999) Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature 397:50–53
Pierres A, Touchard D, Benoliel AM, Bongrand P (2002) Dissecting streptavidin-biotin interaction with a laminar flow chamber. Biophys J 82:3214–3223
Capitanio M, Pavone FS (2013) Interrogating biology with force: single molecule high-resolution measurements with optical tweezers. Biophys J 105:1293–1303
Sarkar R, Rybenkov VV (2016) A guide to magnetic tweezers and their applications. Front Phys 4(48)
Alegre-Cebollada J, Perez-Jimenez R, Kosuri P, Fernandez JM (2010) Single-molecule force spectroscopy approach to enzyme catalysis. J Biol Chem 285:18961–18966
Puchner EM, Gaub HE (2009) Force and function: probing proteins with AFM-based force spectroscopy. Curr Opin Struct Biol 19:605–614
Williams MC, Rouzina I (2002) Force spectroscopy of single DNA and RNA molecules. Curr Opin Struct Biol 12:330–336
Muller DJ, Dufrene YF (2008) Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. Nat Nanotech 3:261–269
Gomez-Moreno C, Lostao A (2013) Single molecule methods to study flavoproteins. In: Hille R, Miller S, Palfey B (eds) Complex flavoproteins, dehydrogenases and physical methods, vol 2. De Gruyter, Boston, pp 277–298
Marcuello C, Arilla-Luna S, Medina M, Lostao A (2013) Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level. Biochim Biophys Acta 1834:665–676
Marcuello C, Arilla-Luna S, Medina M, Lostao A (2012) Atomic force microscopy reveals a dimer of trimers organization in Corynebacterium ammoniagenes FAD synthetase. FEBS J 279:513–514
Ana S, Sebastian M, Arilla-Luna S, Baquedano PMC, Lostao A, Herguedas B, Velázquez-Campoy A, Martínez-Júlvez M, Medina M (2015) Quaternary organization in a bifunctional prokaryotic FAD synthetase: involvement of an arginine at its adenylyltransferase module on the riboflavin kinase activity. Biochim Biophys Acta 1854:897–906
Sebastián M, Lira-Navarrete E, Serrano A, Marcuello C, Velázquez-Campoy A, Lostao A, Hurtado-Guerrero R, Medina M, Martínez-Júlvez M (2017) The FAD synthetase from the human pathogen Streptococcus pneumoniae: a bifunctional enzyme exhibiting activity-dependent redox requirements. Sci Rep 7:7609
Ferreira P, Villanueva R, Martinez-Julvez M, Herguedas B, Marcuello C, Fernandez-Silva P, Cabon L, Hermoso JA, Lostao A, Susin SA, Medina M (2014) Structural insights into the coenzyme mediated monomer-dimer transition of the pro-apoptotic apoptosis inducing factor. Biochemistry 53:4204–4215
Villanueva R, Ferreira P, Marcuello C, Usón A, Miramar MD, Peleato ML, Lostao A, Susin SA, Medina M (2015) Key residues regulating the reductase activity of the human mitochondrial apoptosis inducing factor. Biochemistry 54:5175–5184
Marcuello C, de Miguel R, Gómez-Moreno C, Martínez-Júlvez M, Lostao A (2012) An efficient method for enzyme immobilization evidenced by atomic force microscopy. Protein Eng Des Sel 25:715–723
Marcuello C, de Miguel R, Martínez-Júlvez M, Gómez-Moreno C, Lostao A (2015) Mechanostability of the single electron-transfer complexes of Anabaena ferredoxin–NADP+ reductase. ChemPhysChem 16:3161–3169
Marcuello C, de Miguel R, Martínez-Júlvez M, Gómez-Moreno C, Lostao A (2015) Mechanostability of the single electron-transfer complexes of Anabaena ferredoxin–NADP+ reductase. ChemPhysChem 16(issue 15):3120
Dudko OK, Hummer G, Szabo A (2006) Intrinsic rates and activation free energies from single-molecule pulling experiments. Phys Rev Lett 96:108101
Jarzynski C (1997) Nonequilibrium equality for free energy differences. Phys Rev Lett 78:2690
Tapia-Rojo R, Marcuello C, Gomez-Moreno C, Lostao A, Gómez-Moreno C, Mazo J, Falo F (2015) Characterizing the mechanical dissociation of biological complexes: from forces to free energies. Eur Biophys J 44:130
Tapia-Rojo R, Marcuello C, Lostao A, Gómez-Moreno C, Mazo J, Falo F (2017) A physical picture for mechanical dissociation of biological complexes: from forces to free energies. Phys Chem Chem Phys 19:4567–4575
Marcuello C, de Miguel R, Gomez-Moreno C, Lostao A (2013) Discrimination of protein receptors through quantitative adhesion force maps. Eur Biophys J 42:201
de Pablo PJ, Colchero J, Gomez-Herrero J, Baro AM (1998) Jumping mode scanning force microscopy. Appl Phys Lett 73:3300–3302
Horcas I, Fernández R, Gómez-Rodríguez JM, Colchero J, Gómez-Herrero J, Baro AM (2007) WSxM: a software for scanning probe microscopy and a tool for nanotechnology. Rev Sci Instrum 78:013705
Bouchiat C, Wang MD, Allemand J, Strick T, Block SM, Croquette V (1999) Estimating the persistence length of a worm-like chain molecule from force-extension measurements. Biophys J 76:409–413
Evans E, Ritchie K (1997) Dynamic strength of molecular adhesion bonds. Biophys J 72:1541–1555
Valero-González J, Leonhard-Melief C, Lira-Navarrete E, Jiménez-Osés G, Hernández-Ruiz C, Pallarés MC, Yruela I, Vasudevan D, Lostao A, Corzana F, Takeuchi H, Haltiwanger RS, Hurtado-Guerrero R (2016) A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2. Nat Chem Biol 12:240–246
Pallarés MC, Marcuello C, Botello-Morte L, González A, Fillat MF, Lostao A (2014) Sequential binding of FurA from Anabaena sp. PCC 7120 to iron boxes: exploring regulation at the nanoscale. Biochim Biophys Acta 1844:623–631
Butt HJ, Jaschke M (1995) Calculation of thermal noise in atomic force microscopy. Nanotechnology 6:1–7
Sotres J, Lostao A, Gómez-Moreno C, Baró AM (2007) Jumping mode AFM imaging of biomolecules in the repulsive electrical double layer. Ultramicroscopy 107:1207–1212
Lira-Navarrete E, de Las Rivas M, Compañón I, Pallarés M, Kong Y, Iglesias-Fernández J, Bernardes P, Rovira B, Bruscolini C, Lostao C, Hurtado-Guerrero R (2015) Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation. Nat Commun 6:6937
Wildling L, Unterauer B, Zhu R, Rupprecht A, Haselgrübler T, Rankl C, Ebner A, Vater D, Pollheimer P, Pohl EE, Hinterdorfer P, Gruber HJ (2011) Linking of sensor molecules with amino groups to amino-functionalized AFM tips. Bioconjug Chem 22:1239–1248
Sotres J, Lostao A, Wildling L, Ebner A, Gómez-Moreno C, Gruber HJ, Hinterdorfer P, Baró AM (2008) Unbinding molecular recognition force maps of localized single receptor molecules by atomic force microscopy. ChemPhysChem 9:590–599
Akhremitchev BB (2012) Immobilization and interaction strategies in DFS of biomolecular partners. In: Bizarri AR, Cannistraro S (eds) Dynamic force spectroscopy and biomolecular recognition. CRC Press, Boca Ratón, pp 133–162
Stroh C, Wang H, Bash R, Ashcroft B, Nelson J, Gruber H, Lohr D, Lindsay SM, Hinterdorfer P (2004) Single-molecule recognition imaging microscopy. PNAS 101:12503–12507
Acknowledgments
We thank MINECO (BIO2016-75183-P) and Gobierno de Aragón (E35_17R and LMP58_18) with FEDER (2014-2020) funds for “Building Europe from Aragón” for financial support. A.L. acknowledges support from ARAID. The authors also thank Prof. Carlos Gómez-Moreno for introducing the authors in the flavoproteins field and I. Echániz for technical support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Lostao, A., Medina, M. (2021). Atomic Force Microscopy: Single-Molecule Imaging and Force Spectroscopy in the Study of Flavoproteins Ligand Binding and Reaction Mechanisms. In: Barile, M. (eds) Flavins and Flavoproteins. Methods in Molecular Biology, vol 2280. Springer, New York, NY. https://doi.org/10.1007/978-1-0716-1286-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1286-6_10
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-0716-1285-9
Online ISBN: 978-1-0716-1286-6
eBook Packages: Springer Protocols