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.

Figure 1

Time evolution of ThT fluorescence in HSA solutions incubated at 65°C at pH 7.4 in the (a) absence and (b) presence of 50 mM NaCl.

Figure 2

(a) Far-UV spectra of HSA solutions at 25°C in the presence of 50 mM NaCl at pH 7.4 at 1), 0 h; 2), 24 h; 3), 48 h; 4), 100 h; 5), 200 6), and 7), 250 h of incubation. (b) Far-UV spectra of HSA solutions at 65°C in the presence of 50 mM NaCl at 1), 0 h; 2), 12 h; 3), 24 h; and 4), 48 h of incubation.

Figure 3

Time evolution of secondary structure compositions of HSA solutions at pH 7.4 at 25°C (a and b) or 65°C (c and d) in the absence and presence of 50 mM NaCl, respectively. (▴) α-helix, (■) β-turn, (●) unordered, and (▾) β-sheet conformations.

Figure 4

Second derivative of FTIR spectra at pH 7.4 of (a) native HSA at 25°C before incubation, (b) HSA at 25°C in the presence of 50 mM NaCl, and (c) HSA at 65°C in the absence of electrolyte.

Figure 5

(a) Near-UV CD spectra of HSA solutions at pH 7.4 and 65°C in the absence of electrolyte at 1), 0 h; 2), 6 h; 3), 12 h; 4), 24 h; and 5), 48 h. (b) Time evolution of Trp fluorescence of HSA solutions at 65°C in the (■) absence and (○) presence of 50 mM NaCl.

Figure 6

TEM pictures of the different stages of the HSA fibrillation process at pH 7.4: (a) at 25°C in the presence of 50 mM NaCl after 150 h of incubation, and at 65°C in the presence of 50 mM NaCl after (b) 5 h; (c) and (d) 15 h (part d shows the elongation of oligomers to give bead-like structures); (e) 35 h (where short protofibrils are observed); (f) 45 h; (g) 50 h (where long curly fibrils are seen); and (h–k) after 72 h. Part i shows the addition of oligomers to mature fibrils, j shows the association of mature fibrils in bundles, and k shows mature fibrils with ribbon-like structure.

Figure 7

XRD pattern of HSA fibrils.

Figure 8

Time evolution of ThT fluorescence in HSA solutions incubated at pH 3.0 at 25°C (a and b) or 65°C (c and d) in the absence and presence of 50 mM NaCl, respectively.

Figure 9

Far-UV spectra of HSA solutions at 65°C at pH 3.0 in (a) the absence of electrolyte at 1), 0 h; 2), 24 h; 3), 125 h; 4), 175 h; 5), 200 h; and 6), 250 h of incubation; and (b) the presence of 50 mM NaCl at 1), 0 h; 2), 15 h; 3), 48 h; 4), 100 h; 5), 150 h; and 6), 200 h of incubation.

Figure 10

Time evolution of secondary structure compositions of HSA solutions at pH 3.0 at 25°C (a and b) or 65°C (c and d) in the absence and the presence of 50 mM NaCl, respectively. (▴) α-helix, (■) β-turn, (●) unordered, and (▾) β-sheet conformations.

Figure 11

TEM pictures of the different stages of the HSA fibrillation process at pH 3.0 in the presence of 50 mM NaCl (a) at 25°C after 150 h of incubation, and at 65°C after (b) 24 h, (c) 150 h, and (d) 250 h of incubation in the presence of 50 mM NaCl.

Figure 12

Mechanisms of fibril formation for HSA.