Skip to main content
SpringerLink
Log in

Nanobodies as Structural Probes of Protein Misfolding and Fibril Formation

  • Protocol
  • First Online:
Single Domain Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 911))

Abstract

The deposition of peptides and proteins as amyloid fibrils is a common feature of nearly 50 medical ­disorders affecting the brain or a variety of other organs and tissues. These disorders, which include Alzheimer’s disease, Parkinson’s disease, the prion diseases, and type II diabetes, have an enormous impact on the public health and economy of the modern world. Extensive research is therefore taking place to determine the underlying molecular mechanisms and determinants of the pathological conversion of amyloidogenic proteins from their soluble forms into fibrillar structures. The use of molecular probes and biophysical techniques, such as X-ray crystallography and particularly NMR spectroscopy, are allowing detailed analysis of the mechanism of fibril formation and of the underlying structural and chemical features of the associated pathogenicity. Nanobodies, the antigen-binding domains derived from camelid heavy-chain antibodies, are excellent tools to probe protein aggregation as a result of their exquisite specificity and high affinity and stability, along with their ease of expression and small size; the latter in particular allows them to be used very efficiently in combination with NMR spectroscopy and X-ray crystallography. In this chapter we present an overview of how nanobodies are being used to obtain detailed information on the mechanisms of amyloid formation and on the nature and origin of their links with human diseases.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 75:333–366

    PubMed  CAS  Google Scholar 

  2. Souetre EJ, Qing W, Vigoureux I, Dartigues JF, Lozet H, Lacomblez L, Derouesne C (1995) Economic analysis of Alzheimer’s disease in outpatients: impact of symptom severity. Int Psychogeriatr 7:115–122

    PubMed  CAS  Google Scholar 

  3. Sloane PD, Zimmerman S, Suchindran C, Reed P, Wang L, Boustani M, Sudha S (2002) The public health impact of Alzheimer’s disease, 2000–2050: potential implication of treatment advances. Annu Rev Public Health 23:213–231

    PubMed  Google Scholar 

  4. Small GW, McDonnell DD, Brooks RL, Papadopoulos G (2002) The impact of ­symptom severity on the cost of Alzheimer’s disease. J Am Geriatr Soc 50:321–327

    PubMed  Google Scholar 

  5. Dobson CM (1999) Protein misfolding, ­evolution and disease. Trends Biochem Sci 24:329–332

    PubMed  CAS  Google Scholar 

  6. Dobson CM (2003) Protein folding and ­misfolding. Nature 426:884–890

    PubMed  CAS  Google Scholar 

  7. Luheshi LM, Crowther DC, Dobson CM (2008) Protein misfolding and disease: from the test tube to the organism. Curr Opin Chem Biol 12:25–31

    PubMed  CAS  Google Scholar 

  8. Luheshi LM, Dobson CM (2009) Bridging the gap: from protein misfolding to protein misfolding diseases. FEBS Lett 583:2581–2586

    PubMed  CAS  Google Scholar 

  9. Stefani M, Dobson CM (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med 81:678–699

    PubMed  CAS  Google Scholar 

  10. Vendruscolo M, Zurdo J, MacPhee CE, Dobson CM (2003) Protein folding and ­misfolding: a paradigm of self-assembly and regulation in complex biological systems. Philos Transact A Math Phys Eng Sci 361:1205–1222

    PubMed  CAS  Google Scholar 

  11. Wright CF, Teichmann SA, Clarke J, Dobson CM (2005) The importance of sequence diversity in the aggregation and evolution of proteins. Nature 438:878–881

    PubMed  CAS  Google Scholar 

  12. Ecroyd H, Carver JA (2008) Unraveling the mysteries of protein folding and misfolding. IUBMB Life 60:769–774

    PubMed  CAS  Google Scholar 

  13. Knowles TP, Waudby CA, Devlin GL, Cohen SI, Aguzzi A, Vendruscolo M, Terentjev EM, Welland ME, Dobson CM (2009) An analytical solution to the kinetics of breakable filament assembly. Science 326:1533–1537

    PubMed  CAS  Google Scholar 

  14. Dobson CM (2001) Protein folding and its links with human disease. Biochem Soc Symp 68:1–26

    PubMed  CAS  Google Scholar 

  15. Fandrich M, Fletcher MA, Dobson CM (2001) Amyloid fibrils from muscle myoglobin. Nature 410:165–166

    PubMed  CAS  Google Scholar 

  16. Bucciantini M, Calloni G, Chiti F, Formigli L, Nosi D, Dobson CM, Stefani M (2004) Prefibrillar amyloid protein aggregates share common features of cytotoxicity. J Biol Chem 279:31374–31382

    PubMed  CAS  Google Scholar 

  17. Dumoulin M, Dobson CM (2004) Probing the origins, diagnosis and treatment of amyloid diseases using antibodies. Biochimie 86:589–600

    PubMed  CAS  Google Scholar 

  18. Borrebaeck CA (2000) Antibodies in diagnostics—from immunoassays to protein chips. Immunol Today 21:379–382

    PubMed  CAS  Google Scholar 

  19. Hudson PJ, Souriau C (2003) Engineered antibodies. Nat Med 9:129–134

    PubMed  CAS  Google Scholar 

  20. Valle RP, Jendoubi M (2003) Antibody-based technologies for target discovery. Curr Opin Drug Discov Devel 6:197–203

    PubMed  CAS  Google Scholar 

  21. Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS, Devree BT, Rosenbaum DM, Thian FS, Kobilka TS, Schnapp A, Konetzki I, Sunahara RK, Gellman SH, Pautsch A, Steyaert J, Weis WI, Kobilka BK (2011) Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469:175–180

    PubMed  CAS  Google Scholar 

  22. Baranova E, Fronzes R, Garcia-Pino A, Van Gerven N, Papapostolou D, Pehau-Arnaudet G, Pardon E, Steyaert J, Howorka S, Remaut H (2012) SbsB structure and lattice reconstruction unveil Ca(2+) triggered S-layer assembly. Nature 487(7405):119–122

    PubMed  CAS  Google Scholar 

  23. Muyldermans S, Cambillau C, Wyns L (2001) Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci 26:230–235

    PubMed  CAS  Google Scholar 

  24. Huang L, Muyldermans S, Saerens D (2010) Nanobodies(R): proficient tools in diagnostics. Expert Rev Mol Diagn 10:777–785

    PubMed  Google Scholar 

  25. Revets H, De Baetselier P, Muyldermans S (2005) Nanobodies as novel agents for cancer therapy. Expert Opin Biol Ther 5:111–124

    PubMed  CAS  Google Scholar 

  26. Muyldermans S (2001) Single domain camel antibodies: current status. J Biotechnol 74:277–302

    PubMed  CAS  Google Scholar 

  27. Muyldermans S, Atarhouch T, Saldanha J, Barbosa JA, Hamers R (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 7:1129–1135

    PubMed  CAS  Google Scholar 

  28. Jones DR, Taylor WA, Bate C, David M, Tayebi M (2010) A camelid anti-PrP antibody abrogates PrP replication in prion-permissive neuroblastoma cell lines. PLoS One 5:e9804

    PubMed  Google Scholar 

  29. De Genst EJ, Guilliams T, Wellens J, O’Day EM, Waudby CA, Meehan S, Dumoulin M, Hsu ST, Cremades N, Verschueren KH, Pardon E, Wyns L, Steyaert J, Christodoulou J, Dobson CM (2010) Structure and properties of a complex of alpha-synuclein and a single-domain camelid antibody. J Mol Biol 402:326–343

    PubMed  Google Scholar 

  30. Habicht G, Haupt C, Friedrich RP, Hortschansky P, Sachse C, Meinhardt J, Wieligmann K, Gellermann GP, Brodhun M, Gotz J, Halbhuber KJ, Rocken C, Horn U, Fandrich M (2007) Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Abeta protofibrils. Proc Natl Acad Sci U S A 104:19232–19237

    PubMed  CAS  Google Scholar 

  31. Chan PH, Pardon E, Menzer L, De Genst E, Kumita JR, Christodoulou J, Saerens D, Brans A, Bouillenne F, Archer DB, Robinson CV, Muyldermans S, Matagne A, Redfield C, Wyns L, Dobson CM, Dumoulin M (2008) Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils. Biochemistry 47:11041–11054

    PubMed  CAS  Google Scholar 

  32. Dumoulin M, Canet D, Last AM, Pardon E, Archer DB, Muyldermans S, Wyns L, Matagne A, Robinson CV, Redfield C, Dobson CM (2005) Reduced global cooperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations. J Mol Biol 346:773–788

    PubMed  CAS  Google Scholar 

  33. Dumoulin M, Last AM, Desmyter A, Decanniere K, Canet D, Larsson G, Spencer A, Archer DB, Sasse J, Muyldermans S, Wyns L, Redfield C, Matagne A, Robinson CV, Dobson CM (2003) A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme. Nature 424:783–788

    PubMed  CAS  Google Scholar 

  34. Lafaye P, Achour I, England P, Duyckaerts C, Rougeon F (2009) Single-domain antibodies recognize selectively small oligomeric forms of amyloid beta, prevent Abeta-induced neurotoxicity and inhibit fibril formation. Mol Immunol 46:695–704

    PubMed  CAS  Google Scholar 

  35. Domanska K, Vanderhaegen S, Srinivasan V, Pardon E, Dupeux F, Marquez JA, Giorgetti S, Stoppini M, Wyns L, Bellotti V, Steyaert J (2011) Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic beta2-microglobulin variant. Proc Natl Acad Sci U S A 108:1314–1319

    PubMed  CAS  Google Scholar 

  36. Taguchi H, Planque S, Sapparapu G, Boivin S, Hara M, Nishiyama Y, Paul S (2008) Exceptional amyloid beta peptide hydrolyzing activity of nonphysiological immunoglobulin variable domain scaffolds. J Biol Chem 283:36724–36733

    PubMed  CAS  Google Scholar 

  37. Colby DW, Garg P, Holden T, Chao G, Webster JM, Messer A, Ingram VM, Wittrup KD (2004) Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display. J Mol Biol 342:901–912

    PubMed  CAS  Google Scholar 

  38. Schiefner A, Chatwell L, Korner J, Neumaier I, Colby DW, Volkmer R, Wittrup KD, Skerra A (2011) A disulfide-free single-domain V(L) intrabody with blocking activity towards huntingtin reveals a novel mode of epitope recognition. J Mol Biol 414:337–355

    PubMed  CAS  Google Scholar 

  39. Cookson MR, Xiromerisiou G, Singleton A (2005) How genetics research in Parkinson’s disease is enhancing understanding of the common idiopathic forms of the disease. Curr Opin Neurol 18:706–711

    PubMed  Google Scholar 

  40. Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Hulihan M, Peuralinna T, Dutra A, Nussbaum R, Lincoln S, Crawley A, Hanson M, Maraganore D, Adler C, Cookson MR, Muenter M, Baptista M, Miller D, Blancato J, Hardy J, Gwinn-Hardy K (2003) Alpha-synuclein locus triplication causes Parkinson’s disease. Science 302:841

    PubMed  CAS  Google Scholar 

  41. Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, Stefani M (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416:507–511

    PubMed  CAS  Google Scholar 

  42. Olofsson A, Ostman J, Lundgren E (2002) Amyloid: morphology and toxicity. Clin Chem Lab Med 40:1266–1270

    PubMed  CAS  Google Scholar 

  43. Cremades N, Cohen SI, Deas E, Abramov AY, Chen AY, Orte A, Sandal M, Clarke RW, Dunne P, Aprile FA, Bertoncini CW, Wood NW, Knowles TP, Dobson CM, Klenerman D (2012) Direct observation of the interconversion of normal and toxic forms of alpha-synuclein. Cell 149:1048–1059

    PubMed  CAS  Google Scholar 

  44. Emadi S, Barkhordarian H, Wang MS, Schulz P, Sierks MR (2007) Isolation of a human single chain antibody fragment against oligomeric alpha-synuclein that inhibits aggregation and prevents alpha-synuclein-induced toxicity. J Mol Biol 368:1132–1144

    PubMed  CAS  Google Scholar 

  45. Emadi S, Kasturirangan S, Wang MS, Schulz P, Sierks MR (2009) Detecting morphologically distinct oligomeric forms of alpha-­synuclein. J Biol Chem 284:11048–11058

    PubMed  CAS  Google Scholar 

  46. Emadi S, Liu R, Yuan B, Schulz P, McAllister C, Lyubchenko Y, Messer A, Sierks MR (2004) Inhibiting aggregation of alpha-synuclein with human single chain antibody fragments. Biochemistry 43:2871–2878

    PubMed  CAS  Google Scholar 

  47. Lynch SM, Zhou C, Messer A (2008) An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity. J Mol Biol 377:136–147

    PubMed  CAS  Google Scholar 

  48. Zhou C, Emadi S, Sierks MR, Messer A (2004) A human single-chain Fv intrabody blocks aberrant cellular effects of overexpressed alpha-synuclein. Mol Ther 10:1023–1031

    PubMed  CAS  Google Scholar 

  49. Bermel W, Bertini I, Felli IC, Lee YM, Luchinat C, Pierattelli R (2006) Protonless NMR experiments for sequence-specific assignment of backbone nuclei in unfolded proteins. J Am Chem Soc 128:3918–3919

    PubMed  CAS  Google Scholar 

  50. Tanha J, Xu P, Chen Z, Ni F, Kaplan H, Narang SA, MacKenzie CR (2001) Optimal design features of camelized human single-domain antibody libraries. J Biol Chem 276:24774–24780

    PubMed  CAS  Google Scholar 

  51. Vranken W, Tolkatchev D, Xu P, Tanha J, Chen Z, Narang S, Ni F (2002) Solution structure of a llama single-domain antibody with hydrophobic residues typical of the VH/VL interface. Biochemistry 41:8570–8579

    PubMed  CAS  Google Scholar 

  52. Vilar M, Chou HT, Luhrs T, Maji SK, Riek-Loher D, Verel R, Manning G, Stahlberg H, Riek R (2008) The fold of alpha-synuclein fibrils. Proc Natl Acad Sci U S A 105:8637–8642

    PubMed  CAS  Google Scholar 

  53. Selkoe DJ (2001) Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev 81:741–766

    PubMed  CAS  Google Scholar 

  54. Kuperstein I, Broersen K, Benilova I, Rozenski J, Jonckheere W, Debulpaep M, Vandersteen A, Segers-Nolten I, Van Der Werf K, Subramaniam V, Braeken D, Callewaert G, Bartic C, D’Hooge R, Martins IC, Rousseau F, Schymkowitz J, De Strooper B (2010) Neurotoxicity of Alzheimer’s disease Abeta peptides is induced by small changes in the Abeta42 to Abeta40 ratio. EMBO J 29:3408–3420

    PubMed  CAS  Google Scholar 

  55. Bertram L, Tanzi RE (2008) Thirty years of Alzheimer’s disease genetics: the implications of systematic meta-analyses. Nat Rev Neurosci 9:768–778

    PubMed  CAS  Google Scholar 

  56. Tanzi RE, Bertram L (2005) Twenty years of the Alzheimer’s disease amyloid hypothesis: a genetic perspective. Cell 120:545–555

    PubMed  CAS  Google Scholar 

  57. Ittner LM, Gotz J (2011) Amyloid-beta and tau—a toxic pas de deux in Alzheimer’s disease. Nat Rev Neurosci 12:65–72

    PubMed  CAS  Google Scholar 

  58. Diana FF, Silva Esteves AR, Oliveira CR, Cardoso SM (2011) Mitochondria: the common upstream driver of abeta and tau pathology in Alzheimer s disease. Curr Alzheimer Res 8(5):563–572

    Google Scholar 

  59. LaFerla FM (2010) Pathways linking Abeta and tau pathologies. Biochem Soc Trans 38:993–995

    PubMed  CAS  Google Scholar 

  60. Reiniger L, Lukic A, Linehan J, Rudge P, Collinge J, Mead S, Brandner S (2011) Tau, prions and Abeta: the triad of neurodegeneration. Acta Neuropathol 121:5–20

    PubMed  CAS  Google Scholar 

  61. Small SA, Duff K (2008) Linking Abeta and tau in late-onset Alzheimer’s disease: a dual pathway hypothesis. Neuron 60:534–542

    PubMed  CAS  Google Scholar 

  62. Blurton-Jones M, Laferla FM (2006) Pathways by which Abeta facilitates tau pathology. Curr Alzheimer Res 3:437–448

    PubMed  CAS  Google Scholar 

  63. LaFerla FM, Oddo S (2005) Alzheimer’s disease: Abeta, tau and synaptic dysfunction. Trends Mol Med 11:170–176

    PubMed  CAS  Google Scholar 

  64. Hutton M, McGowan E (2004) Clearing tau pathology with Abeta immunotherapy—reversible and irreversible stages revealed. Neuron 43:293–294

    PubMed  CAS  Google Scholar 

  65. Seino Y, Kawarabayashi T, Wakasaya Y, Watanabe M, Takamura A, Yamamoto-Watanabe Y, Kurata T, Abe K, Ikeda M, Westaway D, Murakami T, Hyslop PS, Matsubara E, Shoji M (2010) Amyloid beta accelerates phosphorylation of tau and ­neurofibrillary tangle formation in an amyloid precursor protein and tau double-transgenic mouse model. J Neurosci Res 88:3547–3554

    PubMed  CAS  Google Scholar 

  66. St George-Hyslop PH, Petit A (2005) Molecular biology and genetics of Alzheimer’s disease. C R Biol 328:119–130

    PubMed  CAS  Google Scholar 

  67. Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300:486–489

    PubMed  CAS  Google Scholar 

  68. Sachse C, Xu C, Wieligmann K, Diekmann S, Grigorieff N, Fandrich M (2006) Quaternary structure of a mature amyloid fibril from Alzheimer’s Abeta(1–40) peptide. J Mol Biol 362:347–354

    PubMed  CAS  Google Scholar 

  69. Liu R, McAllister C, Lyubchenko Y, Sierks MR (2004) Proteolytic antibody light chains alter beta-amyloid aggregation and prevent cytotoxicity. Biochemistry 43:9999–10007

    PubMed  CAS  Google Scholar 

  70. Taguchi H, Planque S, Nishiyama Y, Symersky J, Boivin S, Szabo P, Friedland RP, Ramsland PA, Edmundson AB, Weksler ME, Paul S (2008) Autoantibody-catalyzed hydrolysis of amyloid beta peptide. J Biol Chem 283:4714–4722

    PubMed  CAS  Google Scholar 

  71. Prager EM, Jolles P (1996) Animal lysozymes c and g: an overview. EXS 75:9–31

    PubMed  CAS  Google Scholar 

  72. Booth DR, Sunde M, Bellotti V, Robinson CV, Hutchinson WL, Fraser PE, Hawkins PN, Dobson CM, Radford SE, Blake CC, Pepys MB (1997) Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 385:787–793

    PubMed  CAS  Google Scholar 

  73. Pepys MB, Hawkins PN, Booth DR, Vigushin DM, Tennent GA, Soutar AK, Totty N, Nguyen O, Blake CC, Terry CJ et al (1993) Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature 362:553–557

    PubMed  CAS  Google Scholar 

  74. Desmyter A, Transue TR, Ghahroudi MA, Thi MH, Poortmans F, Hamers R, Muyldermans S, Wyns L (1996) Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nat Struct Biol 3:803–811

    PubMed  CAS  Google Scholar 

  75. Floege J, Ehlerding G (1996) Beta-2-microglobulin-associated amyloidosis. Nephron 72:9–26

    PubMed  CAS  Google Scholar 

  76. Garrett TP, Saper MA, Bjorkman PJ, Strominger JL, Wiley DC (1989) Specificity pockets for the side chains of peptide antigens in HLA-Aw68. Nature 342:692–696

    PubMed  CAS  Google Scholar 

  77. Jones S, Smith DP, Radford SE (2003) Role of the N and C-terminal strands of beta 2-microglobulin in amyloid formation at ­neutral pH. J Mol Biol 330:935–941

    PubMed  CAS  Google Scholar 

  78. Jahn TR, Parker MJ, Homans SW, Radford SE (2006) Amyloid formation under physiological conditions proceeds via a native-like folding intermediate. Nat Struct Mol Biol 13:195–201

    PubMed  CAS  Google Scholar 

  79. Radford SE, Gosal WS, Platt GW (2005) Towards an understanding of the structural molecular mechanism of beta(2)-microglobulin amyloid formation in vitro. Biochim Biophys Acta 1753:51–63

    PubMed  CAS  Google Scholar 

  80. Ami D, Ricagno S, Bolognesi M, Bellotti V, Doglia SM, Natalello A (2012) Structure, stability, and aggregation of beta-2 microglobulin mutants: insights from a Fourier transform infrared study in solution and in the crystalline state. Biophys J 102:1676–1684

    PubMed  CAS  Google Scholar 

  81. Colombo M, de Rosa M, Bellotti V, Ricagno S, Bolognesi M (2012) A recurrent D-strand association interface is observed in beta-2 microglobulin oligomers. FEBS J

    Google Scholar 

  82. Azinas S, Colombo M, Barbiroli A, Santambrogio C, Giorgetti S, Raimondi S, Bonomi F, Grandori R, Bellotti V, Ricagno S, Bolognesi M (2011) D-strand perturbation and amyloid propensity in beta-2 microglobulin. FEBS J 278:2349–2358

    PubMed  CAS  Google Scholar 

  83. Colombo M, Ricagno S, Barbiroli A, Santambrogio C, Giorgetti S, Raimondi S, Bonomi F, Grandori R, Bellotti V, Bolognesi M (2011) The effects of an ideal beta-turn on beta-2 microglobulin fold stability. J Biochem 150:39–47

    PubMed  CAS  Google Scholar 

  84. Fogolari F, Corazza A, Varini N, Rotter M, Gumral D, Codutti L, Rennella E, Viglino P, Bellotti V, Esposito G (2011) Molecular dynamics simulation of beta(2)-microglobulin in denaturing and stabilizing conditions. Proteins 79:986–1001

    PubMed  CAS  Google Scholar 

  85. Koide S (2009) Engineering of recombinant crystallization chaperones. Curr Opin Struct Biol 19:449–457

    PubMed  CAS  Google Scholar 

  86. Abskharon RN, Soror SH, Pardon E, El Hassan H, Legname G, Steyaert J, Wohlkonig A (2011) Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes. Protein Eng Des Sel 24(9):737–741

    PubMed  CAS  Google Scholar 

  87. Biancalana M, Koide S (2010) Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim Biophys Acta 1804:1405–1412

    PubMed  CAS  Google Scholar 

  88. Ban T, Hamada D, Hasegawa K, Naiki H, Goto Y (2003) Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence. J Biol Chem 278:16462–16465

    PubMed  CAS  Google Scholar 

  89. LeVine H III (1999) Quantification of ­beta-sheet amyloid fibril structures with ­thioflavin T. Methods Enzymol 309:274–284

    PubMed  CAS  Google Scholar 

  90. Rogers DR (1965) Screening for amyloid with the thioflavin-T fluorescent method. Am J Clin Pathol 44:59–61

    PubMed  CAS  Google Scholar 

  91. Klunk WE, Jacob RF, Mason RP (1999) Quantifying amyloid by congo red spectral shift assay. Methods Enzymol 309:285–305

    PubMed  CAS  Google Scholar 

  92. Turnell WG, Finch JT (1992) Binding of the dye congo red to the amyloid protein pig insulin reveals a novel homology amongst amyloid-forming peptide sequences. J Mol Biol 227:1205–1223

    PubMed  CAS  Google Scholar 

  93. Benditt EP, Eriksen N, Berglund C (1970) Congo red dichroism with dispersed amyloid fibrils, an extrinsic cotton effect. Proc Natl Acad Sci U S A 66:1044–1051

    PubMed  CAS  Google Scholar 

  94. Goldsbury C, Baxa U, Simon MN, Steven AC, Engel A, Wall JS, Aebi U, Muller SA (2011) Amyloid structure and assembly: insights from scanning transmission electron microscopy. J Struct Biol 173:1–13

    PubMed  CAS  Google Scholar 

  95. Chen B, Thurber KR, Shewmaker F, Wickner RB, Tycko R (2009) Measurement of amyloid fibril mass-per-length by tilted-beam transmission electron microscopy. Proc Natl Acad Sci U S A 106:14339–14344

    PubMed  CAS  Google Scholar 

  96. Legleiter J, Kowalewski T (2004) Atomic force microscopy of beta-amyloid: static and dynamic studies of nanostructure and its formation. Methods Mol Biol 242:349–364

    PubMed  CAS  Google Scholar 

  97. Ding TT, Harper JD (1999) Analysis of amyloid-beta assemblies using tapping mode atomic force microscopy under ambient conditions. Methods Enzymol 309:510–525

    PubMed  CAS  Google Scholar 

  98. Kowalewski T, Holtzman DM (1999) In situ atomic force microscopy study of Alzheimer’s beta-amyloid peptide on different substrates: new insights into mechanism of beta-sheet formation. Proc Natl Acad Sci U S A 96:3688–3693

    PubMed  CAS  Google Scholar 

  99. Goldsbury C, Kistler J, Aebi U, Arvinte T, Cooper GJ (1999) Watching amyloid fibrils grow by time-lapse atomic force microscopy. J Mol Biol 285:33–39

    PubMed  CAS  Google Scholar 

  100. Serpell LC, Fraser PE, Sunde M (1999) X-ray fiber diffraction of amyloid fibrils. Methods Enzymol 309:526–536

    PubMed  CAS  Google Scholar 

  101. Chiti F, Dobson CM (2009) Amyloid formation by globular proteins under native conditions. Nat Chem Biol 5:15–22

    PubMed  CAS  Google Scholar 

  102. Hohlbein J, Gryte K, Heilemann M, Kapanidis AN (2010) Surfing on a new wave of single-molecule fluorescence methods. Phys Biol 7:031001

    PubMed  Google Scholar 

  103. Rigler R (2010) Fluorescence and single ­molecule analysis in cell biology. Biochem Biophys Res Commun 396:170–175

    PubMed  CAS  Google Scholar 

  104. Joo C, Balci H, Ishitsuka Y, Buranachai C, Ha T (2008) Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem 77:51–76

    PubMed  CAS  Google Scholar 

  105. Michalet X, Weiss S, Jager M (2006) Single-molecule fluorescence studies of protein folding and conformational dynamics. Chem Rev 106:1785–1813

    PubMed  CAS  Google Scholar 

  106. Orte A, Birkett NR, Clarke RW, Devlin GL, Dobson CM, Klenerman D (2008) Direct characterization of amyloidogenic oligomers by single-molecule fluorescence. Proc Natl Acad Sci U S A 105:14424–14429

    PubMed  CAS  Google Scholar 

  107. Narayan P, Orte A, Clarke RW, Bolognesi B, Hook S, Ganzinger KA, Meehan S, Wilson MR, Dobson CM, Klenerman D (2012) The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-beta(1–40) peptide. Nat Struct Mol Biol 19:79–83

    CAS  Google Scholar 

  108. Yengo CM, Berger CL (2010) Fluorescence anisotropy and resonance energy transfer: powerful tools for measuring real time protein dynamics in a physiological environment. Curr Opin Pharmacol 10:731–737

    PubMed  CAS  Google Scholar 

  109. Kenworthy AK (2001) Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24:289–296

    PubMed  CAS  Google Scholar 

  110. Matyus L (1992) Fluorescence resonance energy transfer measurements on cell surfaces. A spectroscopic tool for determining protein interactions. J Photochem Photobiol B 12:323–337

    PubMed  CAS  Google Scholar 

  111. Selvin PR (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 7:730–734

    PubMed  CAS  Google Scholar 

  112. Bieschke J, Giese A, Schulz-Schaeffer W, Zerr I, Poser S, Eigen M, Kretzschmar H (2000) Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets. Proc Natl Acad Sci 97:5468–5473

    PubMed  CAS  Google Scholar 

  113. Cao M, Cao P, Yan H, Ren F, Lu W, Hu Y, Zhang S (2008) Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody. Appl Microbiol Biotechnol 79:423–431

    PubMed  CAS  Google Scholar 

  114. Didier P, Weiss E, Sibler AP, Philibert P, Martineau P, Bigot JY, Guidoni L (2008) Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm. Biochem Biophys Res Commun 366:878–884

    PubMed  CAS  Google Scholar 

  115. Schots A, van der Wolf JM (2002) Green fluorescent protein fluobody immunosensors. Immunofluorescence with GFP-antibody fusion proteins. Methods Mol Biol 183:265–273

    PubMed  CAS  Google Scholar 

  116. Colby DW, Chu Y, Cassady JP, Duennwald M, Zazulak H, Webster JM, Messer A, Lindquist S, Ingram VM, Wittrup KD (2004) Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. Proc Natl Acad Sci U S A 101:17616–17621

    PubMed  CAS  Google Scholar 

  117. Messer A, McLear J (2006) The therapeutic potential of intrabodies in neurologic disorders: focus on Huntington and Parkinson diseases. BioDrugs 20:327–333

    PubMed  CAS  Google Scholar 

  118. Miller TW, Messer A (2005) Intrabody ­applications in neurological disorders: progress and future prospects. Mol Ther 12:394–401

    PubMed  CAS  Google Scholar 

  119. Cardinale A, Biocca S (2008) Combating protein misfolding and aggregation by intracellular antibodies. Curr Mol Med 8:2–11

    PubMed  CAS  Google Scholar 

  120. Butler DC, Messer A (2011) Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments. PLoS One 6:e29199

    PubMed  CAS  Google Scholar 

  121. Butler DC, McLear JA, Messer A (2012) Engineered antibody therapies to counteract mutant huntingtin and related toxic intracellular proteins. Prog Neurobiol 97:190–204

    PubMed  CAS  Google Scholar 

  122. Abulrob A, Sprong H, Van Bergen en Henegouwen P, Stanimirovic D (2005) The blood–brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells. J Neurochem 95:1201–1214

    PubMed  CAS  Google Scholar 

  123. Boado RJ (2008) A new generation of neurobiological drugs engineered to overcome the challenges of brain drug delivery. Drug News Perspect 21:489–503

    PubMed  CAS  Google Scholar 

  124. Muruganandam A, Tanha J, Narang S, Stanimirovic D (2002) Selection of phage-displayed llama single-domain antibodies that transmigrate across human blood–brain barrier endothelium. FASEB J 16:240–242

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Medical Research Council, the Wellcome Trust, the Leverhulme Trust, and Parkinson’s UK.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Cambridge, Cambridge, UK

    Erwin De Genst & Christopher M. Dobson

Authors
  1. Erwin De Genst
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher M. Dobson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Erwin De Genst .

Editor information

Editors and Affiliations

  1. , Molecular & Cellular Interactions, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, 1050, Belgium

    Dirk Saerens

  2. , Molecular & Cellular Interactions, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, 1050, Belgium

    Serge Muyldermans

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

De Genst, E., Dobson, C.M. (2012). Nanobodies as Structural Probes of Protein Misfolding and Fibril Formation. In: Saerens, D., Muyldermans, S. (eds) Single Domain Antibodies. Methods in Molecular Biology, vol 911. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-968-6_34

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-968-6_34

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-967-9

  • Online ISBN: 978-1-61779-968-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation