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The amyloid fibril is an insoluble deposit formed in vivo by many different proteins when they undergo certain structural changes or improper processing (Kim et al. 2004). It has been proven that smaller oligopeptide “cassettes” (5-20 aa) of proteins have the potential to self-assemble and form higher ordered amyloid structures of sheets, fibers, ribbons, and nanotubes at certain physiological conditions (Eisenberg et al. 2000, Lu et al. 2003). Although the exact mechanism for self-assembly yielding amyloid is not yet known, metal binding (Viles et al. 2004) and addition of salts (Goto et al. 2005) have been proven to facilitate this process for several proteins. In particular, it has been found that the SO4-2 anion is quite effective in generating amyloid as it requires the lowest optimum concentration, in comparison to ClO4-, I-, and Cl- at pH 2.5, to promote amyloid formation (Goto et al. 2005). Since the Sup35 protein responsible for prion formation in yeast is known to self-assemble in the cytoplasm of the yeast cell, investigating amyloid production of Sup35 cassettes in the presence of SO4-2 at similar physiological conditions in vitro is of particular interest. Our current results indicate no distinguishable difference between the Circular Dichroism spectra of our particular Sup35 cassette PQGGYQQQFN-NH2 with or without SO4-2. However, several parameters of interest have yet to be explored, such as peptide and sulfate concentration and pH of solution. Further studies of these parameters may provide more insight into the true effect of SO4-2.
Amyloid
• Term referring to fibrillar deposit formed in vivo by various human proteins due to partial unfolding and misassembly
• Observed in several diseases (Alzheimer’s disease, Huntington's disease, and Type II Diabetes Mellitus)
• 5 key properties:
1) fibrils are unbranched
2) range from 60 to 120 Å in diameter
3) increased protease resistance
4) bind Congo Red
5) generate a “cross-β” x-ray diffraction pattern
Sup35
• Misfolded version results in translation proceeding despite the presence of stop codons in the mRNA transcript [PSI+] (Eisenberg et al. 2000)
• N domain is essential in prion formation
- rich in Asparagine (N) and Gultamine (Q) residues
- forms amyloid in vivo (Fujita et al. 2003)
- cassettes form amyloid in vitro: GNNQQNY (Doniach et al. 2005)
Peptide Synthesis: Symphony Quartet Peptide Synthesizer was used to make Sup35 cassette (“Q peptide”)
Peptide Purification: Waters 996 High-Performance Liquid Chromatography (HPLC) machine was used to separate impurities from peptide solution
Peptide Freezing/Solidification: Matrix Assisted Laser Disorption/Ionization (MALDI) Mass Spectrometer was used to detect correct peptide peak & ATR Lyopholizer was used to solidify peptide
1.2 mM Peptide Stock solution: 1.5 mg of Q peptide were dissolved in 525 μl 50mM HEPES (N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid) buffer (pH 7) and 525 μl H2O
Samples each containing total volume of 245 μl and 1:3 (0.33mM/1mM), 1:2 (0.5mM/1mM), 1:1(1mM/1mM), 2:1 (2mM/1mM), and 3:1 (3mM/1mM) [SO4-2] : [Peptide] ratios were prepared
Samples were analyzed via Jasco 810 Circular Dichroism (CD) spectropolarimeter
If characteristic β-sheet signature was observed, Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) would then be used to analyze the shape and dimensions of the amyloid structures.
(1mM Peptide 25 mM HEPES pH 7/ H2O)
• No transition to β-strand was observed with SO4-2
• Further experimentation that isolates and selects for optimum peptide and sulfate concentrations and pH of solution required for self-assembly should be pursued
We would like to thank the Howard Hughes Medical Institute EXROP Program for its financial support in allowing this project to be undertaken.
1. Kim, Y. (2004). Protein and Peptide Letters, 11: 23-28.
2. Eisenberg, D. (2000). PNAS, 98: 2375-80.
3. Lu, K. (2003). JACS, 125: 6391-93.
4. Viles, J. (2004). J. Biol. Chem., 279: 18169-77.
5. Goto, Y. (2005). Biochemistry., 44: 1288-99.
6. University of Stuttgart (2002).
7. Fujita, T. (2003). Cell Struct. Funct., 28: 187-93.
8. Doniach, S. (2005). J. Mol. Biol., 349: 648-58.
9. Forsdyke, D. (2003).
10. Alliance Protein Laboratories (2005).
Self-aggregating proteins (amyloid) are known to be involved in several diseases such as Alzheimer's disease, Parkinson's disease, and Type II Diabetes. Several factors, in particular sulfate salts, have been known to affect the rate of amyloid self-assembly. Sup35, a self-aggregating yeast protein, serves as model for studying amyloid kinetics. Thus, it was hypothesized that the presence of sulfate would accelerate the self-assembly process of Sup35.
Peptide Synthesis (Peptide Synthesizer); Peptide Purification (High Pressure Liquid Chromatography); Peptide Characterization (Matrix-Assisted Laser Disorption/Ionization); Peptide Solidifcation (Lypholizer); Circular Dichroism
peptide structure, amyloid, anions, pH, concentration
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