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X-ray Crystallographic Studies of Oligomers Derived from Amyloidogenic Peptides and Proteins

  • Author(s): Spencer, Ryan Kelly
  • Advisor(s): Nowick, James S
  • et al.

This dissertation describes a new class of macrocylic peptides which I invented as a tool for understanding the folding of beta-sheets and the structures of amyloid oligomers. I designed these macrocylic peptides to incorporate various sequences from amyloidogenic peptides and proteins, to fold to adopt beta-sheets, and to further assemble into oligomers. This dissertation details of their formation of beta-sheets by NMR spectroscopy and oligomer structures determined by X-ray crystallography.

In Chapter 2, I describe the development of a new class of macrocyclic beta-sheets that contains an N -methyl amino acid and an amyloidogenic pentapeptide sequence from Abeta, tau, the B chain of insulin, and prion protein. These N-methylated macrocycles fold to adopt beta-sheets in solution. In Chapter 3, I expand these N-methylated beta-sheet peptides to incorporate heptapeptide sequences from beta-amyloid (Abeta17-36). I used these peptides to study the structures of the Abeta oligomers using X-ray crystallography. In Chapter 4, I explain the techniques that I adopted from protein crystallography to solve the X-ray crystallographic structures of these beta-sheet peptides. In Chapter 5, I describe a range of oligomer structures that are accessible to a macrocycle containing a sequence from beta-2-microglobulin (beta2m63-69) and various N-methyl amino acids. The structures formed by these beta-sheet peptides offer insights into the structures formed by amyloidogenic peptides and proteins in amyloid diseases.

Amyloid diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes share common features of toxic soluble protein oligomers. There are no structures at atomic resolution of oligomers formed by full-length amyloidogenic peptides and proteins, and only a few structures of oligomers formed by peptide fragments. The paucity of structural information provides a fundamental roadblock to understanding the pathology of amyloid diseases and developing preventions or therapies. Here, I describe the developement of a new class of beta-sheet macrocycles to study the structures of oligomers formed by amyloidogenic peptides and proteins. Macrocycles containing heptapepeptide sequences from Abeta17-36 fold to adopt beta-sheets and associate further to form triangular trimers and higher-ordered oligomers. These triangular trimers are unprecedented and represent a new motif that full-length amyloidogenic peptides and proteins may adopt.

Macrocyclic peptides derived from beta2m63-69 form beta-sheets that further associate into hexamers, octamers, and dodecamers: the hexamers are trimers of dimers; the octamers are tetramers of dimers; and the dodecamers contain two trimer subunits surrounded by three pairs of beta-sheets. These structures illustrate a common theme in which dimer and trimer subunits further associate to form a hydrophobic core. The seven X-ray crystallographic structures not only illustrate a range of oligomers that a single amyloidogenic peptide sequence can form, but also how mutation can alter the size and topology of the oligomers. A cocrystallization experiment in which a dodecamer-forming peptide recruits a hexamer-forming peptide to form mixed dodecamers demonstrates that one species can dictate the oligomerization of another. These findings should also be relevant to the formation of oligomers of full-length peptides and proteins in amyloid diseases.

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