Colloidal gels from proteins and peptides - Physical Gels from Biological and Synthetic Polymers - page 271

for both dried native and aggregated protein (irrespective of pH) with only a small change
(sharpening) of this feature on heat treatment. At all pH values studied,

-Lg showed only
limited secondary and tertiary structural changes on aggregation (Gosal et al., 2004b ;
Kavanagh et al., 2000a ), in contrast to previous studies of insulin aggregation where
highly ordered crystalline

β

fibrils were indicated (Clark et al., 1981a ).
Although electron microscopy and AFM have shown quite consistently that heating
globular proteins at low pH gives rise to the

fibrillar structures mentioned above, it is
important to point out that almost all of these studies have been carried out well below the
critical gel concentration c 0 ( Chapter 3 ). Nevertheless some conclusions can be reached.
In one such study, TEM revealed many long extended fibrils (~0.1 - 2 μ m in length). The

width distribution of these fibrils, calculated from negatively stained samples, was

narrowly dispersed, the average being of order 10 nm. The absence of higher-order

bril
structure within them was suggested by the absence of any regular helical twist. Some
heterogeneity was revealed by the presence of much shorter, ' worm-like ' fibrils, these

being typically 100 200 nm in length but very similar in width (Gosal et al., 2004b ).

AFM results on the same materials revealed numerous

-

fibrils, varying in length

from ~0.1 to greater than 1 m, similar to those observed in the EM images. However,

the average height of the members of the major population of long

μ

fibrils was found to

-

be ~3 fibrils were also seen, apparently assembled

from two of the smaller underlying components. Work by Donald and co-workers
(Bromley et al., 2005 ; Domike and Donald, 2009 ; Krebs et al., 2009 ) has investigated
other structural aspects of

4 nm. Avery small number of thicker

-Lg amyloid formation and shown how a very large spher-
ulitic aggregate consisting of radially arranged amyloid

β

fibrils can be formed.
At this point it is worth considering the overall conclusions of all this structural work,
especially studies on

-Lg
occurs as dimers, at pH < 3 as monomers and at intermediate pH in a more complex form,
sometimes referred to as an

β -Lg, regarding the assembly of globular proteins. At pH 7 β

≳

. On being heating to moderate temperatures, the
native structure is in equilibrium with a partially folded intermediate, the so-called molten
globule form, formed prior to such transformation to the so-called

' octamer '

state, which seems to
be unstable and prone to aggregation (Hughes and Dunstan, 2009 ). In the model proposed
by Dobson, protein aggregation/gelation is the reverse of protein folding: native monomer
can interact to form native-like aggregates that can then go on to produce

' H '

β -structured

aggregates, which form bres) (Dobson, 2001 , 2003 ). Gelation at or close

to the isoelectric point is indicative of native-like aggregates, and particulate but non-
coagulated gels contain features of both types. The whole

fibrils (amyloid

field of structural aspects of
protein unfolding and subsequent aggregation is very active; for example, we refer the
reader to the work of Dobson and co-workers (Calamai et al., 2005 ; Chiti and
Dobson, 2006 , 2009 ).

9.4.2 Rheological measurements

Rheological measurements on fibrillar protein gels have been carried out by a number of

groups, including Hermansson and Clark and their respective co-workers. For example,
Gosal et al.( 2004a ) measured β -Lg gels at a range of concentrations with pH at 2 and