Abstract
The formation of amyloid fibrils proceeds via a nucleation-dependent mechanism in which nucleation phase is generally associated with a high free energy resulting in the rate-limiting step. On the basis of this kinetic feature, the nucleation is one of the most crucial phases controlling the pathogenesis of amyloidoses, but little is known about the details of how protein molecules and surrounding environment vary at this stage. Here, we applied near infrared (NIR) spectral monitoring of water structural changes in real time during the nucleation-dependent fibrillation of insulin. Whilst multivariate spectral analysis in the 2050–2350 nm spectral region indicated cross-β formation, characteristic transformations of water structure have been detected in the spectral region 1300–1600 nm corresponding to the first overtone of water OH stretching vibrations. Furthermore, specific water spectral patterns (aquagrams) related to different water molecular conformations have been found along the course of protein nucleation and aggregation. Right in the beginning, dissociation of hydrogen-bonded network in bulk water and coinstantaneous protein and ion hydration were observed, followed by water hydrogen-bonded networks development, presumably forcing the nucleation. These specific transformations of water spectral pattern could be used further as a biomarker for early non-invasive diagnosis of amyloidoses prior to explosive amplification and deposits of amyloid fibrils.
Highlights
Amyloid fibrils are supramolecular protein self-assemblies associated with amyloidoses [1,2,3]
We have applied near infrared (NIR) spectroscopy to monitor water structural changes in real time during the fibrillation reaction of human insulin, a 51-residue hormone protein consisting of two polypeptide chains associated with regulating glucose metabolism
Near-infrared spectral monitoring of fibril formation The spontaneous formation of insulin amyloid fibrils without seeds was performed by heating under acidic conditions [24,25,26]: the human insulin dissolved in 25 mM HCl containing 100 mM NaCl at a concentration of 3.0 mg/ml was used as a sample
Summary
Amyloid fibrils are supramolecular protein self-assemblies associated with amyloidoses [1,2,3]. The lag phase is often monotonous without any prominent protein spectroscopic or thermodynamic signals, and much remains to be elucidated regarding how and when nuclei species which template for fibril growth emerge; oligomer-like intermediates have been captured successfully for some proteins [8,9,10,11], their characterization is often difficult due to low population and details of nucleation are still poorly understood, only with atomic images of protein assembly to form oligomeric intermediates as proposed by computational researches [12,13] To explore this issue, we postulate that the molecular structure of water surrounding proteins in aqueous systems will provide valuable clues as to detailed mechanisms of nucleation. NIR spectroscopy is becoming a powerful analytical technique for diagnosis and for investigation of the rearrangement dynamics of the water molecular network [17]
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