To understand the properties of amyloid fibrils and to obtain a mechanistic interpretation of the multi-staged aggregation process detailed knowledge of the molecular structures of the involved species is inevitable. Studying structures of amyloid cross-β spines at atomistic detail has been impeded by their inherently noncrystalline and insoluble nature, as well as their assembly from high-molecular-weight units [4]. Therefore, standard experimental approaches to structure determination of amyloid fibrils formed by natural proteins are not applicable or provide only limited information. In order to permit the study and systematically dissect the structural, physical, and chemical properties of the complex in vivo aggregation process small in vitro model systems were devised [83–90]. This approach helped to develop an understanding of the more general phenomenon of amyloid deposition by a controlled experimental access to the individual
contributions and delicate balance of interactions [90]. The use and biophysical characterization of model peptides for amyloidogenesis is motivated furthermore by the advantage to investigate sequence determinants and rationally study e.g. mutational effects on fibril formation [21,85, 87].
Moreover, computational approaches have identified consensus aggregation-prone sequence motifs of amyloidogenic proteins, therefore leading to the idea that the amyloidogenicity of a sequence can be strongly localized [17,89,91,92]. Indeed, experimental evidence is compiling that protein unfolding is necessary but not sufficient to promote aggregation. Furthermore, it was found that specific short stretches in a sequence can trigger self-assembly and mediate amyloid formation [92–96]. From experiments on amyloidogenic peptide segments many critical observations regarding the energetics and molecular structures of these systems have been derived [90,97,98]. Therefore, one can argue that short model peptides should be more suitable than full-length proteins to investigate those elements in sequences that favor aggregation.
Combined, these experimental findings underscore the notion of amyloid fibril formation being a universal property of the peptide backbone that depends on external factors and is modulated by sequence characteristics [4,16,21,75,99]. Structure based analysis of amyloidogenic sequence signatures predicted short segments and showed that hexapeptides are able to form amyloid-like fibrils [84,88,100]. The ability of these segments to even force a globular, non-fibrillizing protein into the amyloid state was demonstrated [101].
Steric zipper peptides. Crystal structures for a growing number of such mini-mal peptide sequences provided insight into what could be the general spine organization of amyloid fibrils [82,100,102–104]. A common motif, called a steric zipper, was revealed in all of the crystalline structures. The atomic structures show pairs of elongatedβ-sheets with parallel or antiparallel strand alignment. They are interdigitated such that a high complementarity packing of the side chains is achieved, leading to a tight and dry interface. Despite their fundamental similarity, the structures vary in their basic steric zipper motif. Alternative β-sheet packing arrangements of the same segment, as well as distinct β-sheets, built from different segments of a protein, have been found. It has been argued that this can help to understand the observed polymorphism of amyloid structures on a molecular basis [102]. It has been shown that crystalline and fibrillar amyloid polymorphs share fundamental structural characteristics such as the cross-β diffraction pattern [18,82,103–105]. Furthermore, seeding experiments with crystals from fibril-forming short peptides reduce the lag time for the growth of the full-length parent protein fibrils [82,88]. Nevertheless, the degree of order in the
Figure 1.3: Steric zipper structures of peptide segments from fibril-forming proteins.
Representative steric zipper, pair-of-sheets structures for prion Sup35 (class 1), protein tau (class 1) and insulin (class 7) are shown. The dry interface is between the two sheets, showing the front sheet in silver and the rear sheet in purple. Oxygen atoms are depicted in red and nitrogen atoms in blue, respectively. The steric zipper structures fall in different classes (annotated in parentheses) according to packing orientation of the sheets, as well as their strands. Figure adapted from [82] and [100].
crystalline conformations may not entirely represent those in the amyloid fibrils (e.g. the prominent twist in the fibrilar β-sheet arrangement), as indicated by ssNMR measurements on various crystals and fibrils [103–105]. Although crystals and fibrils often grow together in the same solution [82], the crystallization conditions usually involve several chemical additives to promote crystal formation.
Studying small peptides as simplified model systems for amyloidogenic protein aggregation has led to insights into the underlying universal features of fibril formation and provided high-resolution structures of the fibrillar state.
Theory and computation have facilitated the current understanding of the fundamental biophysical aspects and molecular events in the early stages of amyloidogenic peptide ag-gregation [106,107]. Using small peptide fragments and experimentally aquired structural knowledge computer simulation techniques have provided insight into several questions raised, concerning conformational dynamics and thermodynamics of amyloidogenic peptides as well
as aggregation kinetics of oligomeric structures [106,108–127]. MD simulations appear to be particular suited to probe the formation of oligomeric species in atomistic detail and in-form on the transition pathways between them, on timescales still not amenable to experi-ment. For example, based on simulations of Aβ16−22 peptides [124] a dock-lock mechanism has been proposed to explain the commonly implied nucleated growth process of oligomers and fibrils [52,53,57,128]. The authors provide elaborate insight on how monomeric peptides add (dock) to preformed amyloid seed structures in a diffusion-limited process and integrate (lock) by undergoing a substantial conformational conversion [118,124,129–131]. Furthermore, the properties of small multimeric aggregates (dimers to decamers) of various amyloidogenic peptide sequences have been studied by atomistic simulations and described as partially or-dered, nematic structures, which are subject to rapid fluctuations and large conformational rearrangements [106,108,111,115,120,121,131–133]. The obtained oligomer ensembles were distinct from the monomeric form [108,115,130,134] due to conformational changes associ-ated with an emerging β-sheet structure. These structural transitions come to the expense of intra-peptide interactions [108,120,126] and are accompanied by the desolvation of nonpolar surface [106,108,114,126].