Howard Hughes Molecular Biology Summer Research Program Poster, Austin, TX, August, 1995
Microscopy of Curdlan Structure
Iain M. Cheeseman and R. Malcolm Brown, Jr.
Department of Botany, The University of Texas at Austin, Austin, TX., 78712
| Abstract: The structure of curdlan (a (1,3)-ß-glucan) was examined
using light microscopy, transmission electron microscopy (TEM),
and molecular modeling. Light microscopy revealed a doughnut-shaped
structure for the curdlan granule similar to that of starch.
In order to study the structure of the curdlan molecule itself,
a gel was prepared from the curdlan granule. Although curdlan
is insoluble in neutral water, it dissolves easily in a dilute
alkali solution and forms a gel on heating above 54° C.
Low resolution TEM showed that the gel is composed primarily of
intertwining microfibrils at lower temperatures and composed of
associated microfibrils at higher temperatures. High resolution
TEM determined that the microfibrils are composed of three curdlan
molecules that are associated to form a triple helix, and molecular
models of the triple helix were compared to these structures.
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Introduction:
Curdlan is member of the class of molecules known as (1,3)-ß-glucans.
These polysaccharides are characterized by repeating glucose
subunits joined by a ß linkage between the first and third
carbons of the glucose ring. While the primary structure (Fig.
1) is a long chain, curdlan forms more complex tertiary structures
due to intramolecular and intermolecular hydrogen bonding. (1,3)-ß-glucans
are involved in cell structure and food storage in bacteria, fungi
and higher plants. Curdlan in particular shows strong anti-tumor
properties and has utility as a food additive. A firm understanding
of curdlan structure is needed in order to better understand these
uses. For example, it has been shown that there is no anti-tumor
activity when curdlan assumes a random-coil conformation or is
composed of shorter chains, but is greatly enhanced when the curdlan
is found primarily as a single helix (Saitô et al. 1990).
There is still a great deal of confusion concerning the exact
structure of curdlan, because it occurs in a variety of different
states. In its natural state, curdlan is poorly crystalline and
is found as a granule, much like that of starch. The granule
is insoluble in distilled water, but dissolves easily in a dilute
alkali solution, due to the ionization of hydrogen bonds, and
forms a gel when it is heated above 54° C. The gel is composed
mainly of interacting microfibrils (Å100 Å in diameter)
which are made up of many curdlan molecules. There are several
changes that occur with regards to the gel formation due to differences
in the concentration of sodium hydroxide used and the temperature
at which the gel is prepared. One such change is observed as
the concentration of the sodium hydroxide solution that is used
to dissolve the curdlan is raised from 0.19 M to 0.22 M. While
the gels made with less than 0.19 M sodium hydroxide are made
of curdlan molecules with a more ordered structure, using a concentration
of sodium hydroxide above 0.22 M forces the curdlan to assume
a random helix conformation (Saitô et al. 1977). Additional
irreversible changes occur associated with temperature. Although
curdlan gelation begins at 54° C forming what is termed as
a low set gel, an additional change occurs at 95° C to form
what is termed a high set gel (Stone and Clarke 1992). The high
set gel has the properties of being much stronger and more resilient
than the low set gel. This change is explained by the hypothesis
that microfibrils dissociate at 60° C as the hydrogen bonds
are broken, but then reassociate at higher temperatures as hydrophobic
interactions between the curdlan molecules occurs (Harada et al.
1979). An additional change to an even more ordered form is suggested
in some sources (Harada et al. 1979) as the temperature is raised
above 120° C.
In this study, the many levels of curdlan structure were examined.
The granules and their dissolution in sodium hydroxide (caused
by the disruption of hydrogen bonds) were studied using polarized
light microscopy. Low magnification transmission electron microscopy
(TEM) was used to examine the basic gel structure and the interaction
between the microfibrils. High resolution transmission electron
microscopy (HRTEM) was used to examine the fine structure of the
gel to determine what composes the microfibrils and other parts
of the gel, as well as the structure of the curdlan molecule.
Previously it has been suggested that curdlan can exist as a
triple helix, single helix, single chain, or a random coil. The
results of the HRTEM study were compared with molecular models
created from X-ray diffraction data of the curdlan triple helix.
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