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

Influence of Electrostatic Interactions on the Fibrillation Process of Human Serum Albumin
Introduction
Josué Juárez, Sonia Goy López, Adriana Cambón, Pablo Taboada,* and Víctor Mosquera
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
ReceiVed: March 12, 2009; ReVised Manuscript ReceiVed: May 7, 2009
The fibrillation propensity of the multidomain protein human serum albumin (HSA) has been analyzed under
physiological and acidic conditions at room and elevated temperatures with varying ionic strengths by different
spectroscopic techniques. The kinetics of fibril formation under the different solution conditions and the
structures of resulting fibrillar aggregates were also determined. In this way, we have observed that 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
shielding and further enhancement of the solution hydrophobicity might involve a change in the energy
landscape of the aggregation process, which makes the fibrillation process difficult. In contrast, under acidic
conditions, a continuous progressive enhancement of HSA fibrillation is observed as the electrolyte concentration
in solution increases. Both the distinct ionization and initial structural states of the protein before incubation
may be the origin of this behavior. CD, FT-IR, and tryptophan fluorescence spectra seem to confirm this
picture by monitoring the structural changes in both protein tertiary and secondary structures along the
fibrillation process. On the other hand, the fibrillation of HSA does not show a lag phase except at pH 3.0 in
the absence of added salt. Finally, differences in the structure of the intermediates and resulting fibrils under
the different conditions are also elucidated by TEM and FT-IR.
Protein aggregation and, particularly, amyloid formation have
received considerable interest in the fields of protein research
and clinical medicine, since growing evidence has accumulated
that these processes are likely to be key issues in the etiology
of various medical disorders such as Alzheimer’s, Parkinson’s,
Huntington’s, and Creutzfeldt-Jakob diseases, type II diabetes,
and cystic fibrosis among others. In fact, about 20 different
known syndromes are associated with the formation of amyloid
fibrils. 1-6 In addition, a number of nondisease associated proteins
have also been found to be able to form ordered cytotoxic
aggregates and amyloid fibrils in Vitro. 7 On the other hand, the
exceptional physical characteristics of the amyloidal protein
state, such as its stability, mechanical strength, and resistance
to degradation, imply that this type of structure possesses a range
of potential technological applications in biotechnology and
materials science. 8,9
A consensus has emerged that the different processes of in
ViVo and in Vitro aggregation might be linked by the implication
of destabilized protein molecules that undergo non-native
backbone-dominated self-assembly as a competing pathway to
native functional folding. 10 However, both folding and protein
aggregation are thought to underlie some common principles
which aim at minimizing the Gibbs energy of destabilized
protein states, such as the saturation of dangling hydrogen
bonds, 11 the reduction of the solvent-accessible surface area, or
the burial of hydrophobic residues. 12 In this way, globular
proteins generally need to be destabilized (e.g., by mutation, 13,14
heat, 15,16 high pressure, 17,18 low pH, 19,20 or organic denaturant 7,15 )
to aggregate rapidly, with fibril formation proceeding from
* Author to whom correspondence should be addressed. E-mail:
pablo.taboada@usc.es. Telephone: 0034981563100, ext. 14042. Fax:
0034981520676.
J. Phys. Chem. B 2009, 113, 10521–10529 10521
extensively or partially unfolded states 3,21 or, in some cases,
from native-like states in which unfolding may initially be
limited and confined to local regions of the protein. 22,23
The physiological importance of human serum albumin as a
carrier protein and blood pressure regulator and its propensity
to easily aggregate in Vitro have become a good model for
protein aggregation studies. As the phenomenon of protein
aggregation appears to reflect certain generic “polymeric”
features of proteins, 24 the studies of the mechanisms of protein
aggregation on model systems are extremely useful for a better
understanding of the molecular mechanisms of disease-associated
amyloidogenesis. Since in living organisms the native
environment of proteins is a complex composition of water,
cosolvents, and cosolutes which affect the stability of the native
protein fold, 25 experimental approaches to mimic various cellular
environments in Vitro to allow investigations on protein folding
and aggregation often utilize the addition of cosolvents and
cosolutes. Thereby, differences in the properties of the solvent
might induce solvent-adapted structural “responses” of the
aggregating protein. In particular, the importance of electrostatic
interactions in the formation of amyloid fibrils 20,26,27 and the
relevance of the total net charge on the protein to the fibril
formation propensity have been pointed out. 26,27
To put more light on this issue, in the present work we present
a systematic study on the relationship between serum albumin
amyloid-like fibrillation kinetics, fibrillation pathways, and the
resulting aggregated protein structures under different conditions
where the amyloid fibrils of this model protein were found by
changing solution pH and solution ionic strength. 28,29 In this
manner, a modulation of the electrostatic interactions between
protein molecules and aggregates results in different balances
of intermolecular forces between protein molecules, which
involve changes in both protein aggregation and their kinetics.
In this way, we have found large differences in the fibrillation
10.1021/jp902224d CCC: $40.75 © 2009 American Chemical Society
Published on Web 07/02/2009
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