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

Additional Supra-Self-Assembly of Human Serum Albumin under Amyloid-Like-Forming
Solution Conditions
Introduction
Josué Juárez, † Pablo Taboada,* ,† Sonia Goy-López, † Adriana Cambón, †
Marie-Beatrice Madec, ‡ Stephen G. Yeates, ‡ 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, and
Organic Materials InnoVation Center, School of Chemistry, UniVersity of Manchester, Manchester M13 9PL,
United Kingdom
ReceiVed: May 5, 2009; ReVised Manuscript ReceiVed: June 22, 2009
Protein aggregation has a multitude of consequences ranging from affecting protein expression to its implication
in different diseases. Of recent interest is the specific form of aggregation leading to the formation of amyloid
fibrils, structures associated with diseases such as Alzheimer’s disease. These fibrils can further associate in
other more complex structures such as fibrillar gels, plaques, or spherulitic structures. In the present work,
we describe the physical and structural properties of additional supraself-assembled structures of human serum
albumin under solution conditions in which amyloid-like fibrils are formed. We have detected the formation
of ordered aggregates of amyloid fibrils, i.e., spherulites which possess a radial arrangement of the fibrils
around a disorganized protein core and sizes of several micrometers by means of polarized optical microscopy,
laser confocal microscopy, and transmission electron microscopy. These spherulites are detected both in solution
and embedded in an isotropic matrix of fibrillar gels. In this regard, we have also noted the formation of
protein gels when the protein concentration and/or ionic strength exceds a threshold value (the gelation point)
as observed by rheometry. Fibrillar gels are formed through intermolecular nonspecific association of amyloid
fibrils at a pH far away from the isolectric point of the protein where protein molecules seem to display a
“solid-like” behavior due to the existence of non-DLVO (Derjaguin-Landau-Verwey-Overbeck) intermolecular
repulsive forces. As the solution ionic strength increases, a coarsening of this type of gel is observed
by environmental scanning microscopy. In contrast, at pH close to the protein isoelectric point, particulate
gels are formed due to a faster aggregation process, which does not allow substantial structural reorganization
to enable the formation of ordered structures. This behavior also additionally corroborates that the existence
of particulates might also be a generic property of all polypeptide chains as amyloid fibril formation under
suitable conditions.
A number of neurodegenerative diseases have been related
to protein aggregation and deposition in the form of amyloid
fibrils, such as type II diabetes, Alzehimer’s, Parkinson’s,
Huntington’s, and prion diseases amongst others. 1-6 A wide
range of proteins is known to misfold and aggregate in mildly
or hard denaturating conditions as amyloid fibrils provided that
the ability to fibrillate is independent of the original native
structure of the protein, whose amino acid sequence primarily
appears to play a key role in terms of filament arrangement, 7
fibrillation kinetics, 8 and overall yield and stability of the
fibrils. 9,10 These fibrils have been found to be based on a
common structural motif consisting of continuous intermolecular
-sheets which run along the fibril axis such that the individual
-strands are perpendicular to the fibril axis. 11
Under certain conditions, protein fibrils can further associate
either nonspecifically such as in fibrillar gels or plaques or in a
more ordered fashion such as in spherulites. 12-18 It is known
that fibrillar gels are formed by protein aggregation if the protein
concentration exceeds a given critical value under conditions
* Author to whom correspondence should be addressed. E-mail:
pablo.taboada@usc.es.Phone:0034981563100,Ext.14042.Fax:0034981520676.
† Universidad de Santiago de Compostela.
‡ University of Manchester.
J. Phys. Chem. B 2009, 113, 12391–12399 12391
where protein molecules are partially denaturated (as at high
temperatures) and electrically charged (this is, far away from
their isolectric point) upon incubation, i.e., under conditions
where fibrillation can occur. Nevertheless, the correspondence
between fine strand gels and amyloid fibril structure was
established recently. 19,20 In contrast, by decreasing the intermolecular
repulsion through shifting the solution pH to values
near the protein isoelectric point, or by increasing the solution
ionic strength the gel networks are comprised of particulate
aggregates.
On the other hand, for systems where fibrillar protein aggregates
and gels are formed, the strands can further associate in a more
ordered fashion such as spherulitic structures. These are characterized
by the presence of anisotropic material ordered in a spherically
symmetric way. This gives rise to the appearance of a Maltese
cross extinction pattern when these structures are studied under a
polarized light microscope, much in the same way as spherulites
formed by synthetic polymers, 21,22 natural polymers, and biopolymers.
23-25 In solutions in Vitro, spherulites have been reported for
different synthetic and natural peptides, 17,26-30 and have also usually
been associated to the amyloid-like formation pathways of
several proteins such as -lactoglobulin, 20,31,32 R-L-iduronidase, 33
lysozyme, 18 and insulin. 16,34 Spherulites were also found in ViVo
in Alzheimer’s disease, Gerstmann-Straulsler-Scheinker dis-
10.1021/jp904167e CCC: $40.75 © 2009 American Chemical Society
Published on Web 08/14/2009
175