Self-Assembly of Synthetic and Biological Polymeric Systems of ...

5
10
15
20
25
30
35
40
45
Soft Matter
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
Dynamic Article Links ►
PAPER
Hydration effects on the fibrillation process of a globular protein: The
case of human serum albumin.
Josué Juárez, Manuel Alatorre-Meda, Adriana Cambón, Antonio Topete, Silvia Barbosa, Pablo
Taboada*, and Víctor Mosquera.
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
DOI: 10.1039/b000000x
In this work, we have studied the fibrillation process of human serum albumin (HSA) under different
solution conditions. In particular, aggregation kinetics, fibril morphology, and composition structural
changes were investigated at varying experimental conditions such as pH (2.0 and 7.4), temperature (at 25
and 65 ºC), and solvent polarity (ethanol/water mixtures, 10 – 90% v/v). The characterization was carried
out by means of static and dynamic light scattering (SLS and DLS), ThT fluorescence, circular dichroism
(CD) and Fourier Transform Infrared spectroscopies (FT-IR), and transmission electron microscopy
(TEM). The aggregation process and the α-helix to β-sheet transitions were found to be favored by
temperature and physiological pH. Also, pH was observed to influence both the fibrillation pathway and
aggregation kinetics, changing from a classical fibrillation process with a lag phase under acidic
conditions to a downhill polymerization process at physiological pH in the presence of the alcohol.
Regarding protein structural composition, at room temperature and physiological pH ethanol was found to
promote an α-helix to β-sheet conformational transition at intermediate alcohol concentrations, whereas at
low and high ethanol contents α-helix prevailed as the predominant structure. Under acidic conditions,
ethanol promotes an important fibrillation at high cosolvent concentrations due to screening of electric
charges and a decrease in solvent polarity. On the other hand, important differences in the morphology of
the resulting fibrils and aggregates are observed depending on the solution conditions. In particular, the
formation of classical amyloid-like fibrils at physiological pH and high temperature are observed, in
contrast to the usual curly morphology displayed by HSA fibrils under most of solution conditions.
Although high temperature and pH are the main parameters influencing the protein structure
destabilization and subsequent aggregation upon incubation, ethanol helps to regulate the hydrogen
bonding, the attractive hydrophobic interactions, and the protein accessible surface area, thus, modifying
packing constraints and the resulting aggregate morphologies.
Introduction
From a general perspective, amyloid fibril formation present
pros and cons regarding different approaches such as human
health or technological development. On one hand, amyloid
fibrils are recognized as high-performance protein nanomaterials
with a formidable rigidity;
This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 1
1-2 on the other, amyloidosis, the
clinical condition in which amyloid fibrils form from innocuous
soluble proteins, has been found to be involved in diverse
pathological responses as observed in various neurodegenerative
disorders including Alzheimer’s, Parkinson’s, and prion diseases
amongst others. 3-5 applications in the area of nanobiotechnology.
Concerning protein folding and its repercussion on amyloid
fibril formation, it has been demonstrated that fibrillation may
proceed from different protein conformations including
completely/partially unfolded
The elucidation of the underlying molecular
mechanisms for fibril formation would result not only in the
development of strategies for the treatment of amyloidosis-related
disorders but also in the design of new biomaterials for future
Grupo de Física de Coloides y Polímeros, Departamento de Física de la
Materia Condensada, Facultad de Física, Universidad de Santiago de
Compostela, E-15782. Santiago de Compostela. Spain.
pablo.taboada@usc.es
6-11 or fully folded states, 12,13
55
although the essential nature of the resulting amyloid fibrils can
be largely independent of the conformational properties of the
soluble precursors. 6
In living organisms, the native environment of proteins is a
60 complex composition of water, cosolvents and cosolutes which
affect the stability of the native protein fold. 14 In this way,
different experimental approaches using the addition of
cosolvents and cosolutes to mimic various cellular environments
in vitro have provided further knowledge on protein folding and
aggregation/fibrillation mechanisms. 15-20 65
Thereby, cosolventmediated
systems have revealed that differences in solvent
185
50