Protein Structure And Function Flashcards
what is protein denaturation with example conditions?
Protein conformation depends not only on amino acid sequence, but also upon the environment the polypeptide inhabits, both physical and chemical.
If pH, salt concentration, temperature, pressure, etc. are altered, the protein may unravel and loose its native conformation i.e. It becomes denatured.
Extreme heat
Change in pH
Detergents
Urea and guanidine hydrochloride
Exposure to alcohol
Heavy metals
Reducing agents
Change from aqueous to organic solvent such as chloroform
All lead to loss of 3D structure or changes to functional residues (e.g. amino acids in an enzymes active site) so the protein can no longer function properly.
How does temperature cause denaturation?
Heat increases kinetic energy, causes molecule to vibrate enough that H-bonds and hydrophobic interactions are disrupted, and conformation is destabilised.
How does pH change cause denaturation?
Side groups change electrostatic charge so bonds which maintain the protein’s shape (e.g. ionic bonds and H-bonds) are lost.
Most amino acid side chains are not affected by changes in pH.
Some are; those with:
acidic (H+ donating) groups
basic (H+ accepting) groups
Changing pH shifts the equilibrium of ionisation of these acidic and basic groups.
Acid-induced unfolding usually pH 2 – pH 5, base-induced unfolding usually pH 10+.
At close to neutral pH, amino acids exist as zwitterions (NH3+ and CO2- in charged states).
Strong acidic conditions – carboxylate group becomes protonated (NH3+ – CHR – COOH).
Strong basic conditions – ammonio group becomes deprotonated (NH2 – CHR – COO-).
What are the two results of pH change?
- Inactive but still folded
Protein keeps its 3D structure but no longer works well.
e.g. an enzyme might no longer catalyse a reaction because a group that needs to be charged is now uncharged.
Change in catalytic activity with pH can give clues to which amino acids are important in the active site. - Unfolded and inactive
The altered pattern of charges has disrupted the weak, non-covalent forces which stabilise the protein’s 3D structure.
Protein chain unfolds (partially or completely).
Loss of 3D structure renders the protein inactive.
Sometimes pH-induced denaturation can be reversed by restoring the pH to normal.
How do reducing agents cause denaturation?
Disrupt disulphide bonds by adding hydrogen atoms to create sulphydryl moiety (-SH).
Chemical reducing agents include DTT (dithiothreitol) and β-mercaptoethanol.
These can also prevent oxidation and protect correctly formed disulphide bonds within a protein.
Mentioned earlier, chemical reducing agents (e.g. DTT (dithiothreitol), β-mercaptoethanol, glutathione) can disrupt/prevent sulphide bond formation.
Each have their own –SH groups.
Therefore, protect thiol groups in the protein (keep them reduced) as the chemicals are oxidised instead.
What is proteolysis and the inhibitors?
Proteolysis occurs due to exposure to proteolytic enzymes within cells. When cells are lysed, proteases are released compartments (e.g. vacuoles in plants, lysosomes in mammalian cells). When working with proteins, steps must be taken to minimise damage to proteins of interest.
Chemicals which slow down or prevent protease action can be used to help prevent proteolysis.
Generally, a protease inhibitor cocktail is used, which contains a mixture of inhibitors rather than just one.
So that as many different proteases as possible are inhibited; one inhibitor won’t work for all proteases.
Chemicals uses may include:
PMSF (phenylmethyl sulfonyl fluoride) (inhibits variety of proteases).
Pepstatin A (inhibits acid proteases).
Benzamidine (inhibits serine proteases).
Other than inhibitors, how else can protease damage be limited?
Working at reduced temperatures (4 °C) is recommended when working with proteins.
Low temperature reduces the enzymatic activity of any proteases present.
Working as quickly as possible is also advised.
This reduced the time your proteins of interest are exposed to protease activity.
How do you store proteins?
Specific storage required to preserve native protein structures and functionality.
Frozen to prevent gradual decay which would still occur at 4 °C.
Material is usually flash frozen in liquid nitrogen (-196 °C) and stored long-term at -80 °C (or for shorter periods at -20 °C).
Material such as glycerol (up to 50%) is normally added as a cryoprotectant.
Glycerol forms strong hydrogen bonds to slow down water movement, preventing damaging ice crystal formation.
Ice crystals grow, cellular structures get disrupted (releasing more degradative enzymes for example), salts start to crystallize out.
Salts result in a shift in pH (pH problem can be tackled by adding an appropriate buffer to stabilise).
Examples of specificity between enzymes
Hexokinase from the glycolytic pathway catalyses the phosphorylation of glucose.
Glucose Glucose-6-phosphate (reaction uses ATP and releases ADP).
Hexokinase will also catalyse phosphorylation of several other sugars including fructose and mannose.
Urease on the other hand is only specific for urea.
Enzymes with a broader specificity (e.g. hexokinase) have more flexible active site requirements so can accept a wider range of substrate molecules.
Induced fit model
Active site not rigid.
When a substrate binds to the active site, a temporary conformational change in the protein occurs.
The shapes of the active site only becomes complementary to the substrate after it has bound.
Enzymes show a high affinity for the substrate.
The change in shape of the active site orientates the substrate(s) in the right way for the reaction.
Lock and key hypothesis
The substrate of an enzyme has a shape which is complementary in shape to that of the active site.
Explains binding specificity, but not all mechanics of interaction.
Interactions in the binding sites
Substrate binding within an active site can involve a range of chemical bonds e.g.
Hydrophobic interactions.
Weak non-covalent bonds such as hydrogen bonds, ionic bonds, Van der Waals forces.
Structural method of identifying amino acids present in binding site
X-ray crystallography
Involves bombarding a crystal of the protein with X-rays, then analysing the resultant diffraction pattern caused by atoms in the protein scattering the X-rays producing an electron density map.
This map is used to determine the location of atoms relative to each other, bond lengths, and bond angles. You can use this to create a model of the crystal structure. Knowledge of the protein’s primary structure is required to fit the sequence to the 3D map produced.
Chemical methods of determining amino acids present in binding sites
pH Dependence of Activity
Changing pH will affect any amino acids with acidic or basic side chains.
Can potentially be used to investigate the roles of His, Arg, Gln, Glu and Asp amino acids.
e.g. Aspartate has an acidic side group: -COOH -COO- + H+.
Dissociation constant (pKa) for reaction is 3.65, so at pH 3.65 half aa side chains will be ionised and half unionised.
However, at pH 7.65 (physiological) aa is almost 100% ionised.
Using information in reverse can illustrate how pH can be used to locate an aspartate residue in a binding site.
How is aspartate transcarbamoylase an allosteric enzyme?
ATCase is negatively regulated by CTP (cytosine triphosphate).
Presence of CTP means there is no need to make any more.
Negative feedback control / end product inhibition.
CTP acting as an allosteric inhibitor.
CTP binds to regulatory (effector) subunits of the protein, altering the protein structure so the active site formed by the catalytic sub-units is inaccessible.
ATCase is positively regulated by ATP.
ATP is abundant in cells actively making DNA and RNA for growth so it is vital that more pyrimidines (e.g. CTP) are made available to match the availability of the purines (A + T).
