Structure of Self-Assembled Globular Proteins - Food Colloids: Self-Assembly and Material Science - page 38

3.2 Structure of Dilute Aggregates
Individual aggregates can be visualized using microscopy and hence their
degree of connectivity (branching) can be appreciated. However, the estab-
lished techniques of transmission electron microscopy (TEM) and scanning
electron microscopy (SEM) necessitate treatments of the sample that can
potentially perturb the structure. Cryo-TEM does not need such treatment,
but for this technique very thin liquid films are formed on a grid, and the
resulting confinement, shear stress, and interfacial effects could potentially
modify the structure. Light microscopy and confocal laser scanning microscopy
(CLSM) are less perturbing, but only features on length-scales larger than
about a micrometre can be resolved. Atomic force microscopy (AFM) has also
been used; it has very high resolution, but the aggregates need to be adsorbed
on a solid surface, which can again potentially modify the structure. A disad-
vantage of microscopy in general is that a 2-D slice or projection of a 3-D
system is obtained, although SEM does give some impression of the 3-D
structure and CLSM could in principle yield the full 3-D structure.
Very different geometries have been observed using microscopy ranging from
rigid rods to densely branched clusters. Figure 2 shows some examples of
different kinds of b-lactoglobulin (b-LG) aggregates. Rigid rods are formed
under certain conditions when the protein is highly charged and the ionic
strength is low. 11-14 Nevertheless, the rigidity of these aggregates cannot be
explained solely on the basis of electrostatic repulsion because the screening
length of the electrostatic interaction is not much larger than the diameter of
individual proteins. The rods have diameters of a few nanometres, i.e., close to
that of the individual protein molecules; and they may become micrometres
long. Parallels have been drawn between these rod-like aggregates and the
amyloid fibrils that are responsible for a range of diseases. 15 While the mech-
anism of formation is not yet elucidated, it has been speculated that it occurs by
a nucleation and growth process, on the basis of the observation that a few long
rods may be formed in the presence of a large majority of unaggregated
protein. 14,16
Under other conditions, where repulsive electrostatic interactions are still
important, flexible linear aggregates are formed. In these cases the aggregation
Figure 2 Cryo-TEM images of b -LG aggregates formed under different conditions: (a)
pH ¼ 2, no added salt; (b) pH ¼ 2, 0.1 M NaCl; and (c) pH ¼ 7, 0.1 M NaCl.
The total width of each image is 1 m m. (Taken from ref. 17.)