Amyloid Fibrils of Glucagon Characterized by High-Resolution Atomic Force Microscopy

Abstract

Glucagon solutions at pH 2.0 were subjected to mechanical agitation at 37 degrees C in the presence of a hydrophobic surface to explore the details of aggregation and fiber formation. High-resolution intermittent-contact atomic force microscopy performed in solution revealed the presence of aggregates after 0.5 h; however, longer agitation times resulted in the formation of fibrillated structures with varying levels of higher-order assembly. Height, periodicity, and amplitude measurements of these structures allowed the identification of four distinct fiber types. The most elementary fiber form, designated a filament, self-associates in a specific wound fashion to produce protofibrils composed of two filaments. Subsequent self-assembly of these filaments and protofibrils leads to two well-defined fibrillar motifs, termed Type I and Type II. Atomic force microscopy imaging of pH 2.8 glucagon solutions not agitated or exposed to elevated temperature revealed the presence of amorphous aggregates before the formation of fibrillar structures similar to those seen at pH 2.0. Time-course solution Fourier transform infrared spectroscopy and thioflavin T binding studies suggested that glucagon aggregation and fibril formation were associated with the development of beta-sheet structure. The results of these studies are used to describe a possible mechanism for glucagon aggregation and fibrillation that is consistent with a hierarchical assembly model proposed for amyloid fibril formation.

Nanomechanics of individual amyloid fibrils using atomic force microscopy

“…From the images two main types of fibers can be easily identified. One type is thin and low (noted as TA) with height about (2.3±0.3) nm, which is in agreement with the height of profibril as reported in [15]. The other type is thicker and shorter with twisted ribbonlike structures (noted as TB).…”

“…The reasons may go as follows: TB fibril is formed by the intertwining of two (or more) TA fibrils as reported in ref. [15]. The peak of TB fibril is overlapping of two TA fibrils (Figure 3(b)), and there might be some space as inferred from TEM images (Figure 4) between these TA fibrils that may lead to its softness [22].…”

“…On the other hand, amyloid fibers could be potential building blocks for nanomaterials [10]; therefore, exploring their mechanical properties is also beneficial to the final design and fabrication of nanodevices [11,12]. Glucagon is a 29 amino *Corresponding author (email: zhouxingfei@nbu.edu.cn) acid residue peptide which plays an important role in maintaining normal circulating glucose level, and has a strong propensity to aggregate and form fibrils under various conditions [13][14][15]. It is an excellent model molecule for studying protein aggregation and its mechanical properties.…”

Revealing molecular-level surface structure of amyloid fibrils in liquid by means of frequency modulation atomic force microscopy

“…AFM has been used for studying the structure and formation of amyloid fibrils [18][19][20][21]. The highest resolution images have shown protofibril arrangement in a matured fibril with ~10 nm resolution [19], and surface corrugations of polypeptide oligomers with ~3 nm resolution [20].…”

Structural Transitions and Interactions in the Early Stages of Human Glucagon Amyloid Fibrillation

“…The design of aggregation-resistant glucagon analogs has been shown to be beneficial in some cases (11,12). Methionine (Met- 27) oxidation has been shown to greatly delay the fibrillation of unmodified glucagon (10), suggesting that mutations near the C-terminus may be particularly effective. PEGylation has enhanced the physical stability of glucagon under various conditions (13).…”