description of protein stability and the interactions between them and measuring techniques of protein stability

1. Protein stability
D.INDRAJA

2. Protein stability
• Protein stability is the net balance of forces, which determine whether a
protein will be in its native folded conformation or a denatured state.
• Protein stability normally refers to the physical (thermodynamic) stability,
not the chemical stability.
• Structural stability of protein is largely understood by studying various
structures and conformations of protein
Chemical Stability
 Chemical stability involves loss of integrity due to bond cleavage.
 deamination of asparagine and/or glutamine residues,
 hydrolysis of the peptide bond of Asp residues at low pH,
 oxidation of Met at high temperature,
 elimination of disulfide bonds
 disulfide interchange at neutral pH
 Other processes include thiol-catalyzed disulfide interchange and oxidation
of cysteine residues.

3. Measuring protein stability
• The net stability of a protein is defined as the difference in free energy
between the native and denatured state:
• Both GN and GU contribute to G
• It is measuring the energy difference between the U (unfolded) and F
(folded) states.
• The average stability of a monomeric small protein is about kcal/mol, which
is very small!
• ΔDG = GN - GU = -RTlnK
• K=e-ΔG/RT = e-10x1000/(2x298) =2x 10 7
• i.e. in aqueous solution, at room temperature, the ratio of folded : unfolded
protein is 2x 10 7 : 1
• Decreasing the energy of the folded state or increasing the energy of the
unfolded state have the same effect on ΔG.

4. Protein Stability - Importance
• Protein stability is important for many reasons:
• Providing an understanding of the basic thermodynamics of the process of
folding.
• Increased protein stability may be a value in food and drug processing, and in
biotechnology and protein drugs.
• Treatments and drugs that can specifically induce and sustain a strong
chaperone and protease activity in cells
Factors Affecting Protein Stability
1) pH: proteins are most stable in the vicinity of their isoelectric point, pI. In
general, electrostatic interactions are believed to contribute to a small
amount of the stability of the native state; however, there may be
exceptions.
2) Ligand binding: It has been known for a long time that binding ligands, e.g.
inhibitors to enzymes, increases the stability of the protein. This also
applies to ion binding --- many proteins bind anions in their functional
sites.
3) Disulfide bonds: It was observed that many extracellular proteins contained
disulfide bonds; whereas intracellular proteins usually did not exhibit
disulfide bonds.

5. • For many proteins, if their disulfides are broken (i.e. reduced) and then
carboxymethylated with iodoacetate, the resulting protein is denatured, i.e.
unfolded, or mostly unfolded
• Disulfide bonds are believed to increase the stability of the native state by
decreasing the conformational entropy of the unfolded state due to the
conformational constraints imposed by cross-linking (i. e. decreasing the
free energy of the unfolded state).
• Not all residues make equal contributions to protein stability. In fact, it
makes sense that interior ones, inaccessible to the solvent in the native state,
should make a much greater contribution than those on the surface, which
will also be solvent accessible in the unfolded state

7. Protein stability associated functions
Controlling the stability of cellular proteins is a fundamental way by
which cells
• regulate growth,
• differentiation,
• survival, and
• development.
• Measuring the turnover rate of a protein is often the first step in
assessing whether or not the function of a protein is regulated by
proteolysis under specific physiological conditions.

8. Factors affecting protein stability in cell
• A network of highly conserved molecular chaperones and chaperone-related
proteases controls the fold-quality of proteins in the cell.
• Most molecular chaperones can prevent protein aggregation by binding
misfolding intermediates (for example, the GroEL/GroES or the
DnaK/DnaJ/GrpE system).

9. • Some molecular chaperones and chaperone-related proteases, such as the
proteosome, can also hydrolyse ATP to forcefully convert stable harmful
protein aggregates into harmless natively refoldable, or protease-degradable,
polypeptides.
• Molecular chaperones and chaperone-related proteases thus control the
delicate balance between natively folded functional proteins and
aggregation-prone misfolded proteins eventually affecting the stability of
protein, which may form during the lifetime and lead to cell death.
• Therapeutic approaches include treatments and drugs that can specifically
induce and sustain a strong chaperone and protease activity in cells and
tissues prone to toxic protein aggregations.
• Molecular chaperones and the proteases are major clearance mechanisms to
remove toxic protein aggregates from cells, delaying the onset and the
outcome of protein-misfolding diseases

10. Techniques
 Protein stability is measured with several methods such as
 Differential Scanning Calorimetry (DSC)
 Pulse-Chase Method
 Circular Dichroism (CD) Spectroscopy
 Fluorescence-based Activity Assays

11. Differential Scanning Calorimetry (DSC)
• This technique has been widely used in characterizing the stability of
proteins in their native form by measuring the amount of heat required to
denature a particular biomolecule (e.g., protein). Generally, molecules with
higher thermal transition midpoint (Tm) are considered more stable than
those with lower transition midpoints.
• Due to its accuracy and high reproducibility, DSC is recognized as the “gold
standard” for thermal stability analysis and can be used in characterizing and
selecting the most suitable proteins in biotherapeutic development as well as
for ligand interaction studies.
• Additionally, DSC offers the following advantages over other protein
stability determination methods:
• Simple sample preparation
• Thermodynamic parameter determination
• Can use solid and liquid samples
• Can be used to detect denaturation temperatures and irreversible-reversible
phase changes

12. Pulse-Chase Method
• Despite the fact that it involves labelling cells with radioactive precursors or
pulse, the Pulse-Chase method has traditionally been the method of choice
for determining protein stability. Many researchers favour this method since
it allows for the accurate measurement of protein half-life and the
determination of its subcellular localization.
• There are at least two popular non-radioactive versions of this method – the
bleach-chase method and the cycloheximide chase method.
• Bleach-chase method. In this method, the protein of interest is fused with a
fluorophore and bleached with a brief pulse of light to produce a
fluorescently and non-fluorescently protein population. The rate of
degradation is correlated with the rate of fluorescence recovery after the
bleaching process.
• Cycloheximide-chase method. Instead of adding radioactive precursors,
cycloheximide is added to inhibit protein synthesis and allow researchers to
observe and assess the degradation of proteins as a function of time

13. Circular Dichroism (CD) Spectroscopy
• CD spectroscopy provides information on the conformation and stability of
proteins by measuring the differences in absorption between left- and right-
handed polarized light resulting from the structural asymmetry of the protein
of interest. While it provides low-resolution information, CD spectroscopy is
commonly used in most laboratories because it only requires a small amount
of sample, is non-destructive and relatively easy to operate.
Fluorescence-based Activity Assays
• Basically, fluorescence-based activity assays can be used to indirectly
measure protein stability and provide information on protein activity and
functionality. However, since it is highly susceptible to artifacts, coming up
with reproducible results can be a challenge. In addition, the dyes used to
track the stability of the proteins can affect the stability of the protein of
interest.