Skip to main content

Advertisement

SpringerLink
Log in

Effect of environmental conditions on aggregation and fibril formation of barstar

  • Article
  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross-β structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-state→fibril type I→fibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1. a
Fig. 2.
Fig. 3a, b.
Fig. 4. a
Fig. 5.
Fig. 6. a
Fig. 7a–d.
Fig. 8.
Fig. 9.

Similar content being viewed by others

Abbreviations

CD:

circular dichroism

DLS:

dynamic light scattering

EM:

electron microscopy

SLS:

static light scattering

SAXS:

small-angle X-ray scattering

SOXS:

solution X-ray scattering

References

  • Agashe VR, Udgaonkar JB (1995) Thermodynamics of denaturation of barstar: evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride. Biochemistry 34:3286–3299

    CAS  PubMed  Google Scholar 

  • Ballew RM, Sabelko J, Gruebele MRA (1996) Observation of distinct nanosecond and microsecond protein folding events. Nat Struct Biol 3:923–926

    CAS  PubMed  Google Scholar 

  • Bitan G, Lomakin A, Teplow DB (2001) Amyloid β-protein oligomerization. J Biol Chem 276:35176–35184

    Article  CAS  PubMed  Google Scholar 

  • Buckle AM, Schreiber G, Fersht AR (1994) Protein–protein recognition: crystal structural analysis of a barnase–barstar complex at 2.0-A resolution. Biochemistry 33:8878–8889

    CAS  PubMed  Google Scholar 

  • Chiti F, Webster P, Taddei N, Clark A, Stefani M, Ramponi G, Dobson CM (1999) Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc Natl Acad Sci USA 96:3590–3594

    CAS  PubMed  Google Scholar 

  • Conway KA, Harper JD, Lansbury PT (2000) Fibrils formed in vitro from α-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid. Biochemistry 39:2552–2563

    CAS  PubMed  Google Scholar 

  • Damaschun G, Damaschun H, Gast K, Zirwer D (1999) Proteins can adopt totally different folded conformations. J Mol Biol 291:715–725

    CAS  PubMed  Google Scholar 

  • Damaschun G, Damaschun H, Fabian H, Gast K, Kröber R, Wieske M, Zirwer D (2000) Conversion of yeast phosphoglycerate kinase into amyloid-like structure. Proteins Struct Funct Gen 39:204–211

    Article  CAS  Google Scholar 

  • Fändrich M, Fletcher MA, Dobson CM (2001) Amyloid fibrils from muscle myoglobin—even an ordinary globular protein can assume a rogue guise if conditions are right. Nature 410:165–166

    Article  PubMed  Google Scholar 

  • Fersht AR (1995) Characterizing transition states in protein folding—an essential step in the puzzle. Curr Opin Struct Biol 5:79–84

    Article  CAS  PubMed  Google Scholar 

  • Fersht AR (1998) Structure and mechanism in protein folding. WH Freeman, New York

  • Fezoui Y, Hartley DM, Walsh DM, Selkoe DJ, Osterhout JJ, Teplow DB (2000) A de novo designed helix-turn-helix peptide forms nontoxic amyloid fibrils. Nat Struct Biol 7:1095–1099

    Article  CAS  PubMed  Google Scholar 

  • Fink AL, Calciano LJ, Goto Y, Kurotsu T, Palleros DR (1994) Classification of acid denaturation of proteins: intermediates and unfolded states. Biochemistry 33:12504–12511

    CAS  PubMed  Google Scholar 

  • Frisch C, Fersht AR, Schreiber G (2001) Experimental assignment of the structure of the transition state for the association of barnase and barstar. J Mol Biol 308:69–77

    Article  CAS  PubMed  Google Scholar 

  • Gast K, Nöppert A, Müller-Frohne M, Zirwer D, Damaschun G (1997) Stopped-flow dynamic light scattering as a method to monitor compaction during protein folding. Eur Biophys J 25:211–219

    Article  CAS  Google Scholar 

  • Golbik R, Fischer G, Fersht AR (1999) Folding of barstar C40A/C82A/P27A and catalysis of the peptidyl–prolyl cis/trans isomerization by human cytosolic cyclophilin (Cyp18). Protein Sci 8:1505–1514

    CAS  PubMed  Google Scholar 

  • Guijarro JI, Sunde M, Jones JA, Campbell ID, Dobson CM (1998) Amyloid fibril formation by an SH3 domain. Proc Natl Acad Sci USA 95:4224–4228

    Article  CAS  PubMed  Google Scholar 

  • Guillet V, Lapthorn A, Hartley R, Mauguen Y (1993) Recognition between a bacterial ribonuclease, barnase, and its natural inhibitor, barstar. Structure 1:165–176

    CAS  Google Scholar 

  • Haass C, Steiner H (2001) Protofibrils, the unifying toxic molecule of neurodegenerative disorders? Nature Neurosc 4:859–860

    Google Scholar 

  • Hartley RW (1989) Barnase and barstar: two small proteins to fold and fit together. Trends Biochem Sci 14:450–454

    Article  CAS  PubMed  Google Scholar 

  • Hoshino M, Katou H, Hagihara Y, Hasegawa K, Naiki, H, Goto Y (2002) Mapping the core of the β2-microglobulin amyloid fibril by H/D exchange. Nat Struct Biol 9:332–336

    Article  CAS  PubMed  Google Scholar 

  • Janowski R, Kozak M, Jankowska E, Grzonka Z, Grubb A, Abrahamson M, Jaskolski M (2001) Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping. Nat Struct Biol 8:316–320

    Article  CAS  PubMed  Google Scholar 

  • Johnson WC Jr (1990) Protein secondary structure and circular dichroism: a practical guide. Proteins 7:205–214

    CAS  PubMed  Google Scholar 

  • Juneja J, Bhavesh NS, Udgaonkar JB, Hosur RV (2002) NMR identification and characterization of the flexible regions in the 160 kDa molten globule-like aggregate of barstar at low pH. Biochemistry 41:9885–9899

    Article  CAS  PubMed  Google Scholar 

  • Kad NM, Thomson NH, Smith DP, Smith DA, Radford SE (2001) beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro. J Mol Biol 313:559–571

    Article  CAS  PubMed  Google Scholar 

  • Khurana R, Udgaonkar JB (1994) Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule. Biochemistry 33:106–115

    CAS  PubMed  Google Scholar 

  • Khurana R, Hate AT, Nath U, Udgaonkar JB (1995) pH dependence of the stability of barstar to chemical and thermal denaturation. Protein Sci 4:1133–1144

    CAS  PubMed  Google Scholar 

  • Khurana R, Gillespie JR, Talapatra A, Minert LJ, Ionescu ZC, Millett I, Fink AL (2001) Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates. Biochemistry 40:3525–3535

    Article  CAS  PubMed  Google Scholar 

  • Koppel DE (1972) Analysis of macromolecular polydispersity in intensity correlation spectroscopy. The method of cumulants. J Chem Phys 57:4814–4820

    CAS  Google Scholar 

  • Kuwajima K (1989) The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins 6:87–103

    CAS  PubMed  Google Scholar 

  • Liu K, Cho HS, Lashuel HA, Kelly JW, Wemmer DE (2000) A glimpse of a possible amyloidogenic intermediate of transthyretin. Nat Struct Biol 7:754–757

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Gotte G, Libonati M, Eisenberg D (2001) A domain-swapped RNase A dimer with implications for amyloid formation. Nat Struct Biol 8:211–214

    Article  CAS  PubMed  Google Scholar 

  • Lubienski MJ, Bycroft M, Freund SM, Fersht AR (1994) Three-dimensional solution structure and 13C assignments of barstar using nuclear magnetic resonance spectroscopy. Biochemistry 33:8866–8877

    CAS  PubMed  Google Scholar 

  • Lumry R, Eyring H (1954) Conformation changes of proteins. J Phys Chem 58:110–120

    CAS  Google Scholar 

  • MacPhee CE, Dobson CM (2000) Formation of mixed fibrils demonstrates the generic nature and potential utility of amyloid nanostructures. J Am Chem Soc 122:12707–12713

    CAS  Google Scholar 

  • McParland VJ, Kalverda AP, Homans SW, Radford SE (2002) Structural properties of an amyloid precursor of β2-microglobulin. Nat Struct Biol 9:326–331

    Article  CAS  PubMed  Google Scholar 

  • Nölting B (1997) The folding pathway of a protein at high resolution from microseconds to seconds. Proc Natl Acad Sci USA 94:826–830

    Article  PubMed  Google Scholar 

  • Nölting B (1999) Protein folding kinetics (biophysical methods). Springer, Berlin Heidelberg New York

  • Nölting B, Golbik R, Fersht AR (1995) Submillisecond events in protein folding. Proc Natl Acad Sci USA 92:10668–10672

    PubMed  Google Scholar 

  • Nölting B, Golbik R, Soler-González AS, Fersht AR (1997) Circular dichroism of denatured barstar suggests residual structure. Biochemistry 36:9899–9905

    Article  PubMed  Google Scholar 

  • Pepys MB (2001) Pathogenesis, diagnosis and treatment of systemic amyloidosis. Philos Trans R Soc Lond B Biol Sci 356:203–210

    Article  CAS  PubMed  Google Scholar 

  • Provencher SW (1982) CONTIN: a general purpose constrained regularization program for inverting noisy linear algebraic and integral equations. Comput Phys Commun 27:229–242

    Article  Google Scholar 

  • Ptitsyn OB (1995) Molten globule and protein folding. Adv Protein Chem 47:83–229

    CAS  PubMed  Google Scholar 

  • Roberts CJ (2003) Kinetics of irreversible protein aggregation: analysis of extended Lumry–Eyring models and implications for predicting protein shelf life. J Phys Chem B 107:1194–1207

    Article  CAS  Google Scholar 

  • Rochet JC, Lansbury PT (2000) Amyloid fibrillogenesis: themes and variations. Curr Opin Struct Biol 10:60–68

    CAS  PubMed  Google Scholar 

  • Schreiber G, Fersht AR (1996) Rapid, electrostatically assisted association of proteins. Nat Struct Biol 3:427–431

    CAS  PubMed  Google Scholar 

  • Schüler J, Frank J, Saenger W, Georgalis Y (1999) Thermally induced aggregation of human transferrin receptor studied by light scattering techniques. Biophys J 77:1117–1125

    PubMed  Google Scholar 

  • Selkoe DJ (2002) Alzheimer's disease is a synaptic failure. Science 298:789–791

    Article  CAS  PubMed  Google Scholar 

  • Shastry MCR, Udgaonkar JB (1995) The folding mechanism of barstar—evidence for multiple pathways and multiple intermediates. J Mol Biol 247:1013–1027

    Article  CAS  PubMed  Google Scholar 

  • Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287:252–260

    Article  CAS  PubMed  Google Scholar 

  • Wong KB, Freund SMV, Fersht AR (1996) Cold denaturation of barstar: 1H, 15N and 13C NMR assignment and characterisation of residual structure. J Mol Biol 259:805–818

    Article  CAS  PubMed  Google Scholar 

  • Xu S, Bevis B, Arnsdorf M (2001) The assembly of amyloidogenic yeast sup35 as assessed by scanning (atomic) force microscopy: an analogy to linear colloidal aggregation? Biophys J 81:446–454

    Google Scholar 

  • Zaidi FN, Nath U, Udgaonkar JB (1997) Multiple intermediates and transition states during protein unfolding. Nat Struct Biol 4:1016–1024

    CAS  PubMed  Google Scholar 

  • Zurdo J, Guijarro JI, Jimenez JL, Saibil HR, Dobson CM (2001) Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain. J Mol Biol 311:325–340

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Da 292/6–2) and by a grant from the Fonds der Chemischen Industrie to G.D.

Author information

Authors and Affiliations

  1. Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, 13125 , Berlin, Germany

    K. Gast, A. J. Modler, H. Damaschun, R. Kröber, G. Lutsch, D. Zirwer & G. Damaschun

  2. Institut für Biologie c/o Max-Delbrück-Centrum für Molekulare Medizin, Humboldt-Universität zu Berlin, Robert-Rössle-Strasse 10, 13125 , Berlin, Germany

    A. J. Modler

  3. Abteilung Enzymologie, Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 , Halle, Germany

    R. Golbik

Authors
  1. K. Gast
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. J. Modler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. Damaschun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. Kröber
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. G. Lutsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. D. Zirwer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. R. Golbik
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. G. Damaschun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to K. Gast.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gast, K., Modler, A.J., Damaschun, H. et al. Effect of environmental conditions on aggregation and fibril formation of barstar. Eur Biophys J 32, 710–723 (2003). https://doi.org/10.1007/s00249-003-0336-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00249-003-0336-5

Keywords

Search

Navigation