When
proteins are exposed to increasing temperature, losses of
solubility or enzymatic activity occurs over a fairly narrow
range. Depending upon the protein studied and the severity
of the heating, these changes may or may not be reversible.
As
the temperature is increased, a number of bonds in the protein
molecule are weakened. The first affected are the long range
interactions that are necessary for the presence of tertiary
structure. As these bonds are first weakened and are broken,
the protein obtains a more flexible structure and the groups
are exposed to solvent. If heating ceases at this stage the
protein should be able to readily refold to the native structure.
As
heating continues, some of the cooperative hydrogen bonds
that stabilize helical structure will begin to break. As these
bonds are broken, water can interact with and form new hydrogen
bonds with the amide nitrogen and carbonyl oxygens of the
peptide bonds.
The
presence of water further weakens nearby hydrogen bonds by
causing an increase in the effective dielectric constant near
them. As the helical structure is broken, hydrophobic groups
are exposed to the solvent.
The
effect of exposure of new hydrogen bonding groups and of hydrophobic
groups is to increase the amount of water bound by the protein
molecules. The unfolding that occurs increase the hydrodynamic
radius of the molecule causing the viscosity of the solution
to increase. The net result will be an attempt by the protein
to minimize its free energy by burying as many hydrophobic
groups while exposing as many polar groups as possible to
the solvent. While this is analogous to what occurred when
the protein folded originally, it is happening at a much higher
temperature. This greatly weakens the short range interaction
that initially direct protein folding and the structures that
occur will often be vastly different from the native protein.
Upon
cooling, the structures obtained by the aggregated proteins
may not be those of lowest possible free energy, but kinetic
barriers will prevent them from returning to the native format.
Any attempt to obtain the native structure would first require
that the hydrophobic bonds that caused the aggregation be
broken.
This
would be energetically unfavorable and highly unlikely. Only
when all the intermolecular hydrophobic bonds were broken,
could the protein begin to refold as directed by the energy
of short range interactions. The exposure of this large number
of hydrophobic groups to the solvent, however, presents a
large energy barrier that make such a refolding kinetically
unlikely.
Exposure
of most proteins to high temperatures results in irreversible
denaturation. Some proteins, like caseins, however, contain
little if any secondary structure and have managed to remove
their hydrophobic groups from contact with the solvent without
the need for extensive structure. This lack of secondary structure
causes these proteins to be extremely resistant to thermal
denaturation.
The
increased water binding noted in the early stages of denaturation
may be retained following hydrophobic aggregations. The loss
of solubility that occurs will greatly reduce the viscosity
to a level below that of the native proteins.
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