Lecture 1: Importance of PNAs

Question 1

How do protein nucleic acid interactions work at a physical level?

Answer 1

• Small changes like hydrogen bonds can make a big difference.
• DNA molecules have very slightly different sequences.
• The differences in binding energy give differences in equilibrium constants.
• Only the start and end point matter thermodynamically.
• Each molecule has free energy of rotation and translation. They are freer to move when they are separate. They lose entropy when they join together. Entropy is around 60 kj/mol.
• We also need to establish electrostatic and Van der Waals interactions. Pattern on hydrogen bonds. Atoms will make VDW interactions with water separately. At the interface, there is a very tight bond. We get a similar amount of VDW. There isn’t much VDW contribution. If surface isn’t complementary, then there will be a cost in VDW.
• Hydrogen bonds give 20 kj/mol in a vacuum. In reality it’s 4-5 kj/mol because amino acids already have bonds with water. The bonds add up.
• Side chain and main chain atoms will be waving around, and they have entropy. However, they will lose this entropy when the protein binds.
• Surface burial is when hydrophobic parts of the protein are buried.
• Just a few hydrogen bonds can have a large effect.

Question 2

What is the difference between affinity and specificity?

Answer 2

Think of a protein A which can bind to B or C.
• You can have a high affinity for multiple proteins. Not specificity.
• You can’t have low affinity and high specificity.
• Complexes have to have high affinity.

Question 3

How do protein-nucleic acid interactions occur at a kinetic level?

Answer 3

We have the rate of association and dissociation. We can use this to find the equilibrium constant.
• kon is controlled by collisions. Collision rate is about 1 x 108 per second. kon tends to be around 1 x 105 to 1 x 107 moles per second.
• koff is controlled by affinity. We want nanomolar affinity. To get this, koff needs to be around 1 x 10-3 per second.

Question 4

What are the chemical components of nucleic acids?

Answer 4

• There are 4 bases. RNA uses U instead of T because of deamination.
• Each base has a specific hydrogen bonding pattern.
• Ribose acts as a connector. It’s moderately hydrophilic. They don’t have much use in recognition.

Question 5

How do proteins recognise nucleic acids?

Answer 5

• The proteins look for phosphates and hydrogens.
• The proteins want to make direct contact with the edges of the bases for hydrogen bonds.
• There is a spectrum of specificity.
• We can have sequence specific proteins. For example, repressors or proteins.
• We can have proteins which aren’t specific at all. For example, polymerase just wants to bind to DNA/RNA in general.

Question 6

What are base stacking energies?

Answer 6

• We can work out the stacking energy based on the combinations of bases.
• CG-GC is the most stable (-61 kj/mol) while AT-TA is the least stable (-16 kj/mol).
• AT-TA is therefore the easiest DNA sequence to distort. It is more stable.
• The shape of the DNA can therefore control specificity (i.e. is it easy to bind). Protein might not need to contact the bases themselves. This is called indirect binding.

Question 7

What is the structure of A DNA?

Answer 7

• Very rare in vivo for DNA.
• More common for RNA.
• Major groove is narrower and deeper.
• Minor groove is wider and shallower
• Direct readout is less likely.

Question 8

What is the structure of B DNA?

Answer 8

B DNA has a major and minor groove.
• DNA and RNA stack bases to remove the hydrophobic areas.
• 10 bases per turn.
• The first crystal structure for B DNA showed that Watson and Crick were correct overall but there are small deviations from the average based on sequence.
• This was discovered in the 1980s when people could synthesise DNA with specific sequences.
• The Watson-Crick model is an average.
• Base pair parameters such as twists and inclinations can lead to distortions.
• Distortions can also be caused base step parameters. Twist can be changes in the angle between two bases. There can also be tilts or rolls between bases.
• These distortions can change the base stacking energies.
• We can work out the stacking energy based on the combinations of bases.
• CG-GC is the most stable (-61 kj/mol) while AT-TA is the least stable (-16 kj/mol).
• AT-TA is therefore the easiest DNA sequence to distort. It is more stable.
• The shape of the DNA can therefore control specificity (i.e. is it easy to bind). Protein might not need to contact the bases themselves. This is called indirect binding.