1) G.N. Ramachandran created the Ramachandran plot in 1963, which is an essential tool for understanding protein structure. The plot analyzes allowed regions of phi and psi dihedral angles in peptide units. 2) Protein stability refers to a protein maintaining its native folded conformation rather than becoming denatured. Stability depends on a balance of forces and is important for protein function. 3) Factors that influence protein stability include pH, ligand binding, disulfide bonds, and interactions within the protein and between the protein and solvent. Chaperone proteins and proteases also help maintain stability in cells.

1. Protein Stability
MOLECULAR BIOLOGY

2. Protein
Synthesis
• There are 2
processes in
protein synthesis.
• Transcription –
Genes on DNA are
transcribed into an
RNA code.
• Translation – RNA
code is used to
make a
polypeptide.

3. Gopalasamudram Narayana Ramachandran, or
G.N. Ramachandran
(8 October 1922 – 7 April 2001) was an Indian physicist
who was known for his work that led to his creation of
the Ramachandran plot for understanding peptide
structure.
The result of his work on structure of proteins in 1962, -
now commonly known as the Ramachandran plot - was
published in the Journal of Molecular Biology in 1963 and
has become an essential tool in the field of protein
conformation.

4. 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

5. 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.

6. 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

7. 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.

8. Factors Affecting Protein Stability
3) Disulfide bonds:
• 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.

9. Forces that stabilize protein structure
• Interactions between atoms within the protein chain
• Interactions between the protein and the solvent

10. Electrostatic accelerated
protein – protein interaction

11. Ion - Ion (Electrostatics) in Proteins
Figure: Denaturation temperature vs pH
for proteins

12. Metal binding - Note the three histidine
coordinating to the zinc

13. Evolution and Protein Stability
• Why are most proteins only stable to the range of 5-15
kcal/mol ?
•REASON
• If a protein accumulates a stabilizing mutation without evolutionary
pressure to keep it stable, it will soon accumulate another destabilizing
mutation.
• If this protein accumulates a destabilizing mutation which compromises its
ability to function, natural selection will rapidly remove it.
• One example suggestive of this is on the mutation of the barnase to a
close homolog called binase (Serrano et al, 1993). These proteins have 17
differences out of 110 amino-acids, and highly superimposable structures.

14. Cartoon of barnase with all the position that are different in
binase coloured green.
Randomly spread throughout the structure.
Sites showing positions which are
different from barnase.

15. Evolution and Protein Stability
• In the research it was observed that each individual mutation was
made from barnase to binase and each of these point mutations
only affected stability by between +1.1 and -1.1 kcal/mol.
• Which suggested that drift from one protein to another has stability
remarkably constant. This is consistent with the hypothesis that
destabilizing mutations are selected against by reversion or
compensation, and stabilizing mutations are not kept, as they are
not necessary for function.
Another reason for the narrow window of stability observed in the binase / barnase
case is that activity and stability are inversely related, that in order for an enzyme to
be an efficient catalyst, it must be flexible, which leads to a decrease in stability.
This view has been proposed based on the observation that enzymes from hyper-
thermophilic organisms have low activity at mesophilic temperatures (e.g. Hensel,
1993).

16. 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.

17. 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).
• Some molecular chaperones and chaperone-related proteases, such as the
proteasome, 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.

18. Protein stability in different organisms
Terminology
ORGANISMS Type of protein stability leading to specialised
function
psychrophile cold-adapted; cold-loving (-2 ºC to ~15ºC)
mesophile grows at moderate temperatures (~15ºC to ~45ºC) and
conditions.
thermophile heat-loving; grows at >45ºC up to ~80ºC
hyperthermophile grows at >80ºC; limit so far is about 115ºC
halophile grows at high in concentrated salts (internal >1M!)
acidophile grows under highly acidic conditions, i. e., ~ pH 0-pH 2)
barophile adapted to high pressures (e.g., underwater)
extremophiles those that grow under extreme conditions