necessary that the protein is physically stable, i.e., that its structural
integrity and homogeneity, and thus its functionality, are maintained over a reasonably long period of time. Physical stability
relates to several distinct physical properties: structural/conformational stability (is there a single conformation of the protein or is
the protein flexible), thermodynamic stability (the ratio of folded to
unfolded protein molecules), thermostability (the resistance of the
structure to the effects of temperature change), chemostability (the
resistance of the structure to the effects of denaturing agents), and
colloidal stability (the resistance to self-aggregation ¼ solubility).
These physical stabilities are often related to each other as aggregation frequently occurs via unfolded or partly unfolded states. In
protein QC, thermostability and colloidal stability are the two
parameters that are most commonly tested.
1.4.1 Thermal Unfolding
Assays
Thermostability is an important feature that should be optimized
to improve the protein behavior in order to facilitate crystallization
or other structural, biophysical, or functional studies [24–26]. The
melting temperature T m (the temperature at which half of the
protein is folded and half is unfolded) is a useful measure of the
thermostability and a characteristic feature of each protein. T m may
be used to guide optimization of buffer/storage conditions, to
improve stability, or to compare the stability of different protein
variants or proteins from different sources. The thermal stability of
a structure and T m can be determined via denaturation experiments, in which the temperature is scanned across a wide range
and an experimental property related to the extent of folding or
unfolding is recorded at the different temperatures. There are a
number of techniques that can be used to determine the T m of the
sample such as near- or far-UV CD spectroscopy that monitors the
loss of tertiary or secondary structure as the signature of unfolding
(Subheading 1.3), differential scanning fluorimetry (DSF) that
monitors changes in the environment of intrinsic or extrinsic fluorophores, and differential scanning calorimetry (DSC) that measures the change in heat capacity. DSF based on both intrinsic
and extrinsic fluorophores requires relatively small amounts of
sample due to the high intrinsic sensitivity of fluorescence measurements and are inexpensive, whereas DSC is the method of choice in
order to characterize a sample thermodynamically. However, one
should keep in mind that thermodynamic parameters can only be
determined if the unfolding reaction is fully reversible. In order to
test the reversibility of denaturation, the sample should be cooled
down following thermal denaturation and heated up again, checking if the measurement can be duplicated. Reversibility is also
desirable for accuracy of measurements, but is not strictly required,
for QC purposes.
DSF of intrinsic fluorophores monitors the changing behavior of
the protein’s fluorescent residues, usually Trp whose fluorescence
16
Bertrand Raynal et al.
integrity and homogeneity, and thus its functionality, are maintained over a reasonably long period of time. Physical stability
relates to several distinct physical properties: structural/conformational stability (is there a single conformation of the protein or is
the protein flexible), thermodynamic stability (the ratio of folded to
unfolded protein molecules), thermostability (the resistance of the
structure to the effects of temperature change), chemostability (the
resistance of the structure to the effects of denaturing agents), and
colloidal stability (the resistance to self-aggregation ¼ solubility).
These physical stabilities are often related to each other as aggregation frequently occurs via unfolded or partly unfolded states. In
protein QC, thermostability and colloidal stability are the two
parameters that are most commonly tested.
1.4.1 Thermal Unfolding
Assays
Thermostability is an important feature that should be optimized
to improve the protein behavior in order to facilitate crystallization
or other structural, biophysical, or functional studies [24–26]. The
melting temperature T m (the temperature at which half of the
protein is folded and half is unfolded) is a useful measure of the
thermostability and a characteristic feature of each protein. T m may
be used to guide optimization of buffer/storage conditions, to
improve stability, or to compare the stability of different protein
variants or proteins from different sources. The thermal stability of
a structure and T m can be determined via denaturation experiments, in which the temperature is scanned across a wide range
and an experimental property related to the extent of folding or
unfolding is recorded at the different temperatures. There are a
number of techniques that can be used to determine the T m of the
sample such as near- or far-UV CD spectroscopy that monitors the
loss of tertiary or secondary structure as the signature of unfolding
(Subheading 1.3), differential scanning fluorimetry (DSF) that
monitors changes in the environment of intrinsic or extrinsic fluorophores, and differential scanning calorimetry (DSC) that measures the change in heat capacity. DSF based on both intrinsic
and extrinsic fluorophores requires relatively small amounts of
sample due to the high intrinsic sensitivity of fluorescence measurements and are inexpensive, whereas DSC is the method of choice in
order to characterize a sample thermodynamically. However, one
should keep in mind that thermodynamic parameters can only be
determined if the unfolding reaction is fully reversible. In order to
test the reversibility of denaturation, the sample should be cooled
down following thermal denaturation and heated up again, checking if the measurement can be duplicated. Reversibility is also
desirable for accuracy of measurements, but is not strictly required,
for QC purposes.
DSF of intrinsic fluorophores monitors the changing behavior of
the protein’s fluorescent residues, usually Trp whose fluorescence
16
Bertrand Raynal et al.