Appearance of annular ring-like intermediates during amyloid fibril formation from human serum albumin.
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.
Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER), Mohali 140306, Punjab, India. mukhopadhyay@iisermohali.ac.in.
Shruti
S
Arya
Arpana
A
Kumari
Vijit
V
Dalal
Mily
M
Bhattacharya
Samrat
S
Mukhopadhyay
D000682:Amyloid / Q000138:chemical synthesis* / Q000737:chemistry*; D002942:Circular Dichroism; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D018625:Microscopy, Atomic Force; D012709:Serum Albumin / Q000737:chemistry*; D013059:Spectrum Analysis, Raman
D000682:Amyloid; D012709:Serum Albumin