The changing face of glucagon fibrillation: structural polymorphism and conformational imprinting.

We have established a time-resolved fluorescence assay to study fibrillation of the 29 residue peptide hormone glucagon under a variety of different conditions in a high-throughput format. Fibrils formed at pH 2.5 differ in fibrillation kinetics, morphology, thioflavin T staining and FTIR/CD spectra depending on salts, glucagon concentration and fibrillation temperature. Apparent fibrillar stability correlates with spectral and kinetic properties; generally, fibrils formed under conditions favourable for rapid fibrillation (ambient temperatures, high glucagon concentration or high salt concentration) appear less thermostable than those formed under more challenging conditions (high temperatures, low glucagon or low salt concentrations). Properties of preformed fibrils used for seeding are inherited in a prion-like manner. Thus, we conclude that the structure of fibrils formed by glucagon is not the result of the global energy minimization, but rather kinetically controlled by solvent conditions and seed-imprinting. Fibrillar polymorphism, which is being reported for an increasing number of proteins, probably reflects that fibrils have not been under evolutionary constraints to retain a single active conformation. Our results highlight the complexity of the fibrillation mechanism of glucagon, since even subtle changes in fibrillation conditions can alter the type of fibrils formed, or result in formation of mixtures of several types of fibrils.

[1]  W. Gratzer,et al.  Conformational states of glucagon. , 1967, Biochemical and biophysical research communications.

[2]  Chan Beum Park,et al.  Insulin amyloid fibrillation at above 100°C: New insights into protein folding under extreme temperatures , 2004 .

[3]  U Aebi,et al.  Polymorphic fibrillar assembly of human amylin. , 1997, Journal of structural biology.

[4]  A. Fersht.  Structure and mechanism in protein science , 1998 .

[5]  Pawel Sikorski,et al.  Molecular basis for amyloid fibril formation and stability. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A model for the three-dimensional structure of glucagon. , 1983, Journal of theoretical biology.

[7]  Jan Vos,et al.  A flexible sigmoid function of determinate growth. , 2003, Annals of botany.

[8]  Narasimha Sreerama,et al.  Structural composition of βI‐ and βII‐proteins , 2003 .

[9]  C. Krittanai,et al.  Correcting the circular dichroism spectra of peptides for contributions of absorbing side chains. , 1997, Analytical biochemistry.

[10]  J. Rumfeldt,et al.  Sonication of proteins causes formation of aggregates that resemble amyloid , 2004, Protein science : a publication of the Protein Society.

[11]  S. Lindquist,et al.  Hsp104 Catalyzes Formation and Elimination of Self-Replicating Sup35 Prion Conformers , 2004, Science.

[12]  Wei Wang,et al.  Protein aggregation and its inhibition in biopharmaceutics. , 2005, International journal of pharmaceutics.

[13]  E. Bradbury,et al.  A conformational study of glucagon. , 1968, European journal of biochemistry.

[14]  A. Fink,et al.  Fourier transform infrared spectroscopy in analysis of protein deposits. , 1999, Methods in enzymology.

[15]  S. Onoue,et al.  Mishandling of the Therapeutic Peptide Glucagon Generates Cytotoxic Amyloidogenic Fibrils , 2004, Pharmaceutical Research.

[16]  T. Ban,et al.  Direct Observation of Amyloid Fibril Growth Monitored by Thioflavin T Fluorescence* 210 , 2003, The Journal of Biological Chemistry.

[17]  R. Wetzel.  For Protein Misassembly, It's the “I” Decade , 1996, Cell.

[18]  C. MacPhee,et al.  Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops. , 2000, Biochemistry.

[19]  F. J. Richards.  A Flexible Growth Function for Empirical Use , 1959 .

[20]  H. Levine.  Stopped-flow kinetics reveal multiple phases of thioflavin T binding to Alzheimer beta (1-40) amyloid fibrils. , 1997, Archives of biochemistry and biophysics.

[21]  C. Dobson.  Protein misfolding, evolution and disease. , 1999, Trends in biochemical sciences.

[22]  M. Davies,et al.  Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate. , 2003, Biochemistry.

[23]  P. Privalov,et al.  Energetics of protein structure. , 1995, Advances in protein chemistry.

[24]  H. Levine.  Quantification of beta-sheet amyloid fibril structures with thioflavin T. , 1999, Methods in enzymology.

[25]  R. Epand.  Cation-induced conformational change in glucagon. , 1982, Molecular pharmacology.

[26]  George B. Benedek,et al.  Temperature dependence of amyloid β-protein fibrillization , 1998 .

[27]  A. Donald,et al.  The binding of thioflavin-T to amyloid fibrils: localisation and implications. , 2005, Journal of structural biology.

[28]  Yuji Goto,et al.  Direct Measurement of the Thermodynamic Parameters of Amyloid Formation by Isothermal Titration Calorimetry* , 2004, Journal of Biological Chemistry.

[29]  D. Otzen,et al.  Correspondence between anomalous m‐ and Δ Cp‐values in protein folding , 2004 .

[30]  U Aebi,et al.  Architecture and polymorphism of fibrillar supramolecular assemblies produced by in vitro aggregation of human calcitonin. , 1995, Journal of structural biology.

[31]  Peter Chien,et al.  Emerging principles of conformation-based prion inheritance. , 2004, Annual review of biochemistry.

[32]  B. Permanne,et al.  Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding , 2001, Nature.

[33]  J. T. Yang,et al.  Helical conformation of glucagon in surfactant solutions. , 1980, Biochemistry.

[34]  D. Otzen,et al.  Designed protein tetramer zipped together with a hydrophobic Alzheimer homology: a structural clue to amyloid assembly. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[35]  P. Lansbury,et al.  Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. , 1997, Annual review of biochemistry.

[36]  D. Otzen,et al.  Directed evolution of barnase stability using proteolytic selection. , 2002, Journal of molecular biology.

[37]  S. Frokjaer,et al.  Studies of the structure of insulin fibrils by Fourier transform infrared (FTIR) spectroscopy and electron microscopy. , 2001, Journal of pharmaceutical sciences.

[38]  P. Lansbury,et al.  Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB. , 1992, Biochemistry.

[39]  P. Roepstorff,et al.  Unfolding, aggregation, and seeded amyloid formation of lysine-58-cleaved beta 2-microglobulin. , 2005, Biochemistry.

[40]  W. C. Johnson,et al.  Secondary structure of proteins through circular dichroism spectroscopy. , 1988, Annual review of biophysics and biophysical chemistry.

[41]  L. Serpell,et al.  Protofilaments, filaments, ribbons, and fibrils from peptidomimetic self-assembly:  implications for amyloid fibril formation and materials science. , 2000, Journal of the American Chemical Society.

[42]  Stanley B. Prusiner,et al.  Nobel Lecture: Prions , 1998 .

[43]  A. Fink,et al.  Spectral Properties of Thioflavin T and Its Complexes with Amyloid Fibrils , 2003 .

[44]  H. M. Petrassi,et al.  An engineered transthyretin monomer that is nonamyloidogenic, unless it is partially denatured. , 2001, Biochemistry.

[45]  V. Uversky,et al.  Effect of Association State and Conformational Stability on the Kinetics of Immunoglobulin Light Chain Amyloid Fibril Formation at Physiological pH* , 2002, The Journal of Biological Chemistry.

[46]  V. Uversky,et al.  Evidence for a Partially Folded Intermediate in α-Synuclein Fibril Formation* , 2001, The Journal of Biological Chemistry.

[47]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[48]  W. Gratzer,et al.  Formation and structure of gels and fibrils from glucagon. , 1969, European journal of biochemistry.

[49]  V. Uversky,et al.  Partially folded intermediates in insulin fibrillation. , 2003, Biochemistry.

[50]  Gerd Buntkowsky,et al.  Solid State NMR Reveals a pH-dependent Antiparallel β-Sheet Registry in Fibrils Formed by a β-Amyloid Peptide , 2004 .

[51]  Jesper Søndergaard Pedersen,et al.  Modulation of S6 fibrillation by unfolding rates and gatekeeper residues. , 2004, Journal of molecular biology.

[52]  R. Turner,et al.  Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[53]  P. V. von Hippel,et al.  Calculation of protein extinction coefficients from amino acid sequence data. , 1989, Analytical biochemistry.

[54]  V. Uversky,et al.  Conformational constraints for amyloid fibrillation: the importance of being unfolded. , 2004, Biochimica et biophysica acta.

[55]  Richard D. Leapman,et al.  Self-Propagating, Molecular-Level Polymorphism in Alzheimer's ß-Amyloid Fibrils , 2005, Science.

[56]  D. Otzen,et al.  Elimination of a misfolded folding intermediate by a single point mutation. , 2004, Biochemistry.

[57]  F. Ferrone,et al.  Analysis of protein aggregation kinetics. , 1999, Methods in enzymology.

[58]  R. Jansen,et al.  Insulin forms amyloid in a strain‐dependent manner: An FT‐IR spectroscopic study , 2004, Protein science : a publication of the Protein Society.

[59]  J. Hofrichter,et al.  Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[60]  J. M. Creeth,et al.  Presence ot trimers in glucagon solution. , 1972, European journal of biochemistry.

[61]  J T Finch,et al.  Amyloid fibers are water-filled nanotubes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[62]  R. Epand,et al.  Evidence for the compact conformation of monomeric glucagon. Hydrogen-tritium exchange studies. , 1972, Biochemistry.

[63]  P. Y. Chou,et al.  Conformational transitions of glucagon in solution: The α → β transition , 1977 .

[64]  D. Patel.  Proton Nuclear Magnetic Resonance Study of Glucagon , 1970 .

[65]  M. Fändrich,et al.  FTIR reveals structural differences between native β‐sheet proteins and amyloid fibrils , 2004, Protein science : a publication of the Protein Society.

[66]  G. Fasman,et al.  Quantitative analysis of cyclic β‐turn models , 1992, Protein science : a publication of the Protein Society.

[67]  P. Callis,et al.  Mechanisms of tryptophan fluorescence shifts in proteins. , 2001, Biophysical journal.

[68]  Christopher M. Dobson,et al.  The protofilament structure of insulin amyloid fibrils , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[69]  A. D. Osborne,et al.  Secondary structure and dynamics of glucagon in solution. , 1982, Biochimica et biophysica acta.

[70]  R. Tycko.  Progress towards a molecular-level structural understanding of amyloid fibrils. , 2004, Current opinion in structural biology.

[71]  D. Otzen,et al.  Using micellar mole fractions to assess membrane protein stability in mixed micelles. , 2005, Biochimica et biophysica acta.

[72]  C. Soto,et al.  Cyclic amplification of protein misfolding: application to prion-related disorders and beyond , 2002, Trends in Neurosciences.

[73]  V. Uversky,et al.  Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. , 2001, Biochemistry.

[74]  D. Hall,et al.  Silent prions lying in wait: a two-hit model of prion/amyloid formation and infection. , 2004, Journal of molecular biology.

[75]  J. Hofrichter,et al.  Rate of Intrachain Diffusion of Unfolded Cytochrome c , 1997 .

[76]  U Aebi,et al.  Amyloid fibril formation from full-length and fragments of amylin. , 2000, Journal of structural biology.

[77]  S. Müller,et al.  Studies on the in Vitro Assembly of Aβ 1–40: Implications for the Search for Aβ Fibril Formation Inhibitors , 2000 .

[78]  B. Chernow,et al.  Glucagon: Hormone or therapeutic agent? , 1984, Critical care medicine.

[79]  G. Glenner,et al.  β-PLEATED SHEET FIBRILS A COMPARISON OF NATIVE AMYLOID WITH SYNTHETIC PROTEIN FIBRILS , 1974, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[80]  Ronald Wetzel,et al.  Seeding Specificity in Amyloid Growth Induced by Heterologous Fibrils* , 2004, Journal of Biological Chemistry.

[81]  R. Wetzel,et al.  Physical, morphological and functional differences between ph 5.8 and 7.4 aggregates of the Alzheimer's amyloid peptide Abeta. , 1996, Journal of molecular biology.

[82]  S. Yadav,et al.  A new method for the determination of stability parameters of proteins from their heat-induced denaturation curves. , 2000, Analytical biochemistry.

[83]  D. Otzen,et al.  Salt-induced detour through compact regions of the protein folding landscape. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[84]  Andrew F. Hill,et al.  Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD , 1996, Nature.