Appearance of annular ring-like intermediates during amyloid fibril formation from human serum albumin

Abstract

The self-assembly of proteins triggered by a conformational switch into highly ordered β-sheet rich amyloid fibrils has captivated burgeoning interest in recent years due to the involvement of amyloids in a variety of human diseases and a diverse range of biological functions. Here, we have investigated the mechanism of fibrillogenesis of human serum albumin (HSA), an all-α-helical protein, using an array of biophysical tools that include steady-state as well as time-resolved fluorescence, circular dichroism and Raman spectroscopy in conjunction with atomic force microscopy (AFM). Investigations into the temporal evolution of nanoscale morphology using AFM revealed the presence of ring-like intermediates that subsequently transformed into worm-like fibrils presumably by a ring-opening mechanism. Additionally, a multitude of morphologically-diverse oligomers were observed on the pathway to amyloid formation. Kinetic analysis using multiple structural probes in-tandem indicated that HSA amyloid assembly is a concerted process encompassing a major structural change that is primarily mediated by hydrophobic interactions between thermally-induced disordered segments originating in various domains. A slower growth kinetics of aggregates suggested that the protein structural reorganization is a prerequisite for fibril formation. Moreover, time-dependent Raman spectroscopic studies of HSA aggregation provided key molecular insights into the conformational transitions occurring within the protein amide backbone and at the residue-specific level. Our data revealed the emergence of conformationally-diverse disulfides as a consequence of structural reorganization and sequestration of tyrosines into the hydrophobic amyloid core comprising antiparallel cross β-sheets.

Optical spectroscopic methods for diagnostics of intervertebral disc degeneration

“…7a), and it is probably related to hormonal change. This result confirms the earlier data that the transforms of amide I include an amyloid formation that causes the amide I band structure to change [31], and more amyloid was observed in the patient over 50 years [32]. The component at 2707 cm -1 showed a better relation to the LOD, as the dependences on LOD were similar to both genders.…”

Detergent-induced aggregation of an amyloidogenic intrinsically disordered protein

“…AFM is used extensively for imaging the nanoscale topography of protein aggregates. 58,[73][74][75][76] In the absence of SDS, we observed long thread-like amyloid fibrils of 6-7 nm in height. The height profiles of these fibrils observed in AFM images collected at different time points in the growth phase of aggregation are shown in Figure 5A-C.…”

“…The concentration of the labeled protein was estimated using ε 337nm = 6100 M −1 cm −1 for AEDANS. 58 The AEDANS labeled κ-casein was stored under denatured condition (6 M GdmCl in pH 7, 50 mM phosphate buffer).…”

Unravelling the fibrillation mechanism of ovalbumin in the presence of mercury at its isoelectric pH

“…Worm like brils normally appear from partially unfolded state of protein via non-nucleated pathway. 26 These are less ordered compared to LS brillar counterparts. Both LS and WL brils can bind to the amyloid-specic dye Congo red and Thioavin-T.…”