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

structure of HSA molecule at physiological solution conditions is composed of 67% -helix, 5%
-sheet, 12% turn, and 17% extended chain (133). The three-dimensional configuration of
serum albumin is divided into three major domains (labelled as I, I and III), and each domain
are composed of two subdomains that possess common structural motifs, which are
predominantly helical and cross-linked by the disulfide bridges (134)(135).
The HSA structure can be influenced by several factors as pH, temperature, cosolvents... It is
known that HSA molecule shows a number of well-defined pH-dependent structural
transitions, (Figure 5.6) (136): E form (below pH 2.7), F form (pH 2.7-4.3), N form (pH 4.3-8), B
form (pH 8-10) and A form (over pH 10) (134)-(137). On the other hand, during its thermal
unfolding process, three states can be distinguished: the native form, the reversible unfolded
form (at 65 0 C) and the irreversible unfolded form (at 74 0 C) (120). Both unfolding processes
give rise to aggregation-prone conformational changes of both secondary and tertiary
structures, allowing the formation of amorphous aggregates, intermediate states, oligomers
and amyloid fibrils.
Due to the physiological importance of human serum albumin as a carrier protein and blood
pressure regulator and its propensity to easily aggregate in vitro, HSA has become a good
model for protein aggregation studies. Moreover, as the phenomenon of protein aggregation
appears to reflect certain generic “polymeric” features of proteins (138), the study of protein
aggregation mechanisms in model systems is extremely useful to gain a better understanding
about the molecular mechanisms of disease-associated amyloidogenesis.
In this way, in this work we show the propensity of HSA to form amyloid fibrils and other types
of aggregates under different thermal and solvent conditions (changing the solution pH,
solution ionic strength and solvent composition), allowing the modulation of electrostatic,
hydrophobic and hydration interactions between protein molecules and aggregates (139)-
(141). The kinetic aggregation, protein conformational changes upon self-assembly, and
structure of the different intermediates on the fibrillation pathway were determined by means
of ThT fluorescence and CR absorbance; far and near-UV-Vis CD; tryptophan fluorescence;
Fourier transform infrared spectroscopy; X-ray diffraction; and TEM, SEM, AFM and optical
microscopy. We have observed that the fibril formation is largely affected by electrostatic
shielding: at physiological pH, fibrillation is progressively more efficient and faster in the
presence of up to 50 mM NaCl; meanwhile, at larger salt concentrations, excessive charge
shielding and further enhancement of the solution hydrophobicity might involve a change in
140