Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

Abstract

The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance; far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the beta-sheet (up to 26%) and unordered conformation at the expense of alpha-helical conformation, as revealed by ThT fluorescence and circular dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long enough for the ends to find themselves regularly in close proximity to each other. These fibrils can be formed by an antiparallel arrangement of beta-strands forming the beta-sheet structure of the HSA fibrils as the most probable configuration. Very long incubation times lead to a more complex morphological variability of amyloid mature fibrils (i.e., long straight fibrils, flat-ribbon structures, laterally connected fibers, etc.). We also observed that mature straight fibrils can also grow by protein oligomers tending to align within the immediate vicinity of the fibers. This filament + monomers/oligomers scenario is an alternative pathway to the otherwise dominant filament + filament manner of the protein fibril's lateral growth. Conformational preferences for a certain pathway to become active may exist, and the influence of environmental conditions such as pH, temperature, and salt must be considered.

What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

“…Therefore, and as suggested by the protein depletion results, amorphous aggregation seems to prevail over amyloid fibrillization from the beginning of incubation. The CD intensity continues to decrease from day 4 to day 7 as a consequence of the formation of off-pathway aggregates and the increased number and size of scattering objects in solution (19). No amyloid fibrils are formed during this period, as indicated by the ThT fluorescence plateau in Fig.…”

“…The hyperbolic (concave) profiles exhibiting no inflection point or lag phase in the absence of seeding (Fig. 1a) are frequently reported in the literature during the aggregation of, for example, serum albumin (19,20), transthyretin (21,22), ␤ 2 -microglobulin (23), the four-repeat domain of Tau (24), apolipoprotein (25), and amyloid-␤ variants (26,27). This type of result is explained by the CLM as the result of predominant primary nucleation over the autocatalytic processes and is also well fitted by the "Ockham's razor" minimalistic model (18,28).…”

A Kinetic Study of Ovalbumin Fibril Formation: The Importance of Fragmentation and End-Joining

“…Fibril polymorphism may become manifest when the self-assembly takes place under different conditions, as seen for b2-microglobulin (18), or, alternatively, two forms may arise simultaneously, as observed for amyloid b (19,20). Intriguingly, a number of systems that form fibers with wormlike chains have also been observed to form ring or loop structures (e.g., apolipoprotein C-II (21), a-synuclein (22), the crystallins (23,24), human serum albumin (25), b-lactoglobulin (26), and a S2 -casein (27)). The simplest hypothesis for the formation of loops by protein fibrils is the joining of their two ends (28).…”

Amyloid fibril formation from human and bovine serum albumin followed by quasi-simultaneous Fourier-transform infrared (FT-IR) spectroscopy and static light scattering (SLS)

“…Structural changes during BSA and HSA fibril formation Human serum albumin (HSA), bovine serum albumin (BSA), and other albumins have been reported to form amyloid fibrils in vitro (Juárez et al 2009;Militello et al 2004;Pearce et al 2007). Although they are not predisposed to form this kind of aggregate, because of the lack of properties that suggest this predisposition and their high a-helix structure content, there are conditions that favor partly destabilized monomers and dimers, for example low pH, high temperatures, or the presence of chemical denaturants, that can force amyloidogenesis (Gorinstein et al 2002).…”