1. Advanced Higher Cells and Proteins
Dna, Proteins and Binding to ligands

2. Think
What proteins are associated with DNA? How are proteins involved in transcription? How is protein production controlled? Why is it important that protein production is controlled? Why is protein structure important in relation to its function?
Think about these questions and answer them as you study this section on protein structure.

3. DNA and proteins This lesson will cover
DNA and its associated proteins
Other proteins involved with transcription

4. DNA and protein association

5. DNA and protein association
DNA binds to a number of proteins.
Positively charged histone proteins bind to the
negatively charged sugar-phosphate backbone of
DNA in eukaryotes.
DNA is wrapped around histones to form nucleosomes
packing the DNA in chromosomes.

6. DNA and protein association
Animation

7. Histone proteins and nucleosome

8. Other DNA proteins and ligand binding
Other proteins have binding sites that are specific to
particular sequences of double stranded DNA.
When this happens they can stimulate or inhibit the
initiation of transcription.
Animation

9. Dna and protein complex in transcription

10. Transcription Factors
Transcription factors (TFs) are molecules involved in regulating gene expression.
They are usually proteins, (they can be short, non-coding RNA).
TFs are also usually found working in groups or complexes, forming multiple interactions that allow for varying degrees of control over rates of transcription.

11. Transcription Factors
In people (and other eukaryotes), genes are usually in a default "off" state, so TFs serve mainly to turn gene expression "on".
TFs work by recognizing certain nucleotide sequences (motifs) before or after the gene on the chromosome.
The TFs bind, attract other TFs and create a complex that eventually facilitates binding by RNA polymerase, thus beginning the process of transcription.

12. Binding changes the conformation of a protein
Proteins including enzymes are three-dimensional and have a specific shape or conformation.
As a ligand binds to a protein binding site, or a substrate binds to an enzyme’s active site, the conformation of the protein changes.
This change in conformation causes a functional change in the protein and may activate or deactivate it.

13. Binding to ligands A ligand is a substance that can bind to a protein.
R groups not involved in protein folding can allow binding to these other molecules.
Binding sites will have complementary shape and chemistry to the ligand.
The ligand can either be a substrate or a molecule that affects the activity of the protein.

14. Enzymes
All chemical reactions require energy to enable them, this is the activation energy.
Enzymes lower the activation energy.
2 types of reaction are:
Anabolic (synthesis) a dehydration synthesis reaction.
Catabolic (degradation) a hydrolysis reaction.

15. Anabolic Reactions
ENDOTHERMIC REACTION
Uses energy to SYNTHESISE large molecules from smaller ones e.g.
Amino Acids Proteins
Also known as endothermic reactions

16. Catabolic Reactions
EXOTHERMIC REACTION
These release energy through the BREAKDOWN of large molecules into smaller units e.g.
Cellular Respiration:
ATP ADP + Pi
Also known as exothermic reactions

17. Enzyme types
Proteases - break down proteins into amino acids by breaking peptide bonds (hydrolysis).
Nucleases - break down nucleic acids into nucleotides (hydrolysis).
Kinases - adds phosphate group to a molecule.
Phosphatases – removes phosphate group
ATPases - hydrolysis of ATP.

18. Control of Enzyme activity
Control of enzyme activity occurs in these ways
number of enzyme molecules present
compartmentalisation
change of enzyme shape by
competitive inhibitors, non-competitive inhibitors,
enzyme modulators, covalent modification
end product inhibition

19. How do enzymes work?

20. Induced fit and enzymes
Enzymes are not necessarily a perfect sit to substrate
The enzyme changes shape in response to close association with the substrate.
This the Induced fit theory

21. Competitive inhibition
A molecule close to shape of substrate competes directly for active site so reducing the concentration of available enzyme.
This can be reversed by increasing the concentration of the correct substrate unless the binding of competitor is irreversible.

22. Malonate is the competitive inhibitor
Malonate example
Succinate dehydrogenase catalyses the oxidation of succinate to fumarate (respiration)
Malonate is the competitive inhibitor

24. Non-competitive inhibition
An inhibitor binds to the enzyme molecule at a different area and changes the shape of the enzyme including the active site.
This may be a permanent alteration or may not.

26. Inhibition can either be reversible or non-reversible
Some inhibitors bind irreversibly with the enzyme molecules.
The enzymatic reactions will stop sooner or later and are not affected by an increase in substrate concentration.
Irreversible inhibitors include heavy metal ions such as silver, mercury and lead ions.

27. Enzyme modulators
Some enzymes change their shape in response to a regulating molecule.
These are called allosteric enzymes
Positive modulators (activators)
stabilise enzyme in the active form.
Negative modulators (inhibitors)
stabilise enzyme in the inactive form.

28. Allosteric Enzymes

29. Covalent modifications
Involves the addition, modification or removal of a variety of chemical groups to or from an enzyme (often phosphate.)
These result in a change in the shape of the enzyme and so its activity.
These include phosphorylation by kinases and dephosphorylation by phosphatases.
Conversion of inactive forms to active forms e.g. trypsinogen and trypsin

30. An example of activation is trypsinogen to trypsin
trypsinogen activated by
enterokinase in duodenum

31. Trypsin is synthesised in the pancreas, but not in its active form as it would digest the pancreatic tissue
Therefore it is synthesised as a slightly longer protein called TRYPSINOGEN
Activation occurs when trypsinogen is cleaved by a protease in the duodenum
Once active, trypsin can activate more trypsinogen molecules

32. End product Inhibition
Often seen in pathways that involve a series of enzyme controlled reactions.
The end product once produced has an inhibiting affect on an enzyme in the reaction.
Example:
Bacterial production of amino acid isoleucine from threonine.
5 stages enzyme controlled
Threonine Isoleucine

33. To summarise As a ligand binds to a protein or a substrate binds to
an enzyme’s active site, the conformation of the
protein changes,
This change in conformation causes a functional
change in the protein.

34. To summarise
In enzymes, specificity between the active site and substrate is
related to induced fit.
When the correct substrate starts to bind, a temporary change in
shape of the active site occurs increasing the binding and interaction with the substrate.
The chemical environment produced lowers the activation energy
required for the reaction.
Once catalysis takes place, the original enzyme conformation is
resumed and products are released from the active site.

35. To summarise
In allosteric enzymes, modulators bind at secondary binding sites.
The conformation of the enzyme changes and this alters the
affinity of the active site for the substrate.
Positive modulators increase the enzyme affinity whereas
negative modulators reduce the enzymes affinity for the substrate.

36. Haemoglobin and Oxygen

37. Cooperativity in hemoglobin
Deoxyhaemoglobin has a relatively low affinity for oxygen. As one molecule of oxygen binds to one of the four haem groups in a hemoglobin molecule it increases the affinity of the remaining three haem groups to bind oxygen. Conversely, oxyhaemoglobin increases its ability to loose oxygen as oxygen is released by each successive haem group. This creates the classic sigmoid shape of the oxygen dissociation curve.

38. Dissociation curve of haemoglobin
Rightward shifts in the dissociation curve indicate decreased affinity of haemoglobin for oxygen, making it harder to bind the oxygen in the lungs but easier to release oxygen to respiring tissues. A higher partial pressure of oxygen is therefore required in the lungs for uptake of oxygen by deoxyhaemoglobin.
Leftward shifts indicate increased affinity, making it easier to bind oxygen in the lungs but increasing the difficulty to release oxygen to respiring tissues. A lower partial pressure of oxygen is therefore required by respiring tissues to release the oxygen from oxyhaemoglobin.

39. Disassociation releasing oxygen to tissues
Deoxyhaemoglobin
Oxyhaemoglobin
Association binding oxygen in lungs

40. Effects of temperature and pH
Low pH = low affinity.
High temperature = low affinity.
Exercise increases body temperature and produces more
CO2, acidifying the blood.
This has a corresponding effect on the oxyhaemoglobin
dissociation curve.

42. Sickle Cell Anaemia Low oxygen levels cause change in haemoglobin
structure.
Strands cause cells to take
on bent sickle shape
blocking capillaries.

43. High Altitude and Oxygen
The concentration of oxygen (O2) in sea-level air is 20.9%, so the partial pressure of O2 (pO2) is kPa. Atmospheric pressure decreases exponentially with altitude while the O2 fraction remains constant to about 100 km, so pO2 decreases exponentially with altitude as well. It is about half of its sea-level value at 5,000 m (16,000 ft), the altitude of the Everest Base Camp, and only a third at 8,848 m (29,029 ft), the summit of Mount Everest. When pO2 drops, the body responds with altitude acclimatization.

44. Dissociation curve of haemoglobin
Rightward shifts in the dissociation curve indicate decreased affinity of haemoglobin for oxygen, making it harder to bind the oxygen in the lungs but easier to release oxygen to respiring tissues. A higher partial pressure of oxygen is therefore required in the lungs for uptake of oxygen by deoxyhaemoglobin.
Leftward shifts indicate increased affinity, making it easier to bind oxygen in the lungs but increasing the difficulty to release oxygen to respiring tissues. A lower partial pressure of oxygen is therefore required by respiring tissues to release the oxygen from oxyhaemoglobin.

45. To summarise
Some proteins with quaternary structure show cooperativity
in which changes in binding alter the affinity of the remaining
subunits.
Cooperativity exists in the binding and release of oxygen in
Haemoglobin.
Temperature and pH influence oxygen association.