Oxford Biochemistry interview questions: the role of energy

When answering a biological problem, the default is to resort to something like: a protein folds because it has a function, or the RNA binds because the gene needs to be transcribed. But this is almost a tautology. Biochemistry is about explaining biology at the cellular and molecular level. And biology is subject to the laws of chemistry, meaning we have to consider the role of energy.

Explaining the role of energy in biochemical processes is likely to come up as an Oxford biochemistry interview question.

Let's take a look at 3 examples.

1. Protein folding

2. Transcription

3. ATP hydroysis

Protein folding

A protein folds because it is energetically favourable to do so. What does this mean?

All molecules have free energy, and a molecule is more stable the lower its free energy is. We can't measure absolute energy, but we can measure energy changes. The change in free energy is ΔG.

When talking about free energy change, we are talking about free energy change of the system. Proteins do not fold in a vacuum; they fold in water. The protein and the water is the system.

Most proteins have a reasonably large proportion of hydrophobic R groups. In the unfolded state, these R groups cannot make hydrogen bonds with water, which means that enthalpy is not as low as it could be (forming covalent bonds / intermolecular forces RELEASES energy).

Likewise, the protein is unfolded, occupying a large SA:V ratio, and because most of that surface area is hydrophobic, the water molecules have to fit around it awkwardly to maintain their own internal hydrogen bonding network. This awkward fit reduces the number of combinations in space of hydrogen bonding of water: reduced entropy.

R: R groups; Blue: hydrophillic; Red: hydrophobic

So, by folding up, the R groups are tucked away from the water and can form stronger Van Der Waals interactions with each other in the core of the protein (reduced enthalpy). Protein folding means it has a lower SA:V volume ratio so the entropy of the water is less impacted (increased entropy). Of course, as a protein folds, its own entropy goes down - it goes from flexible conformations to a defined tertiary structure.

So really, protein folding is all about the entropy of water

Some proteins, however, don't fold. They stay unfolded. These proteins have a high proportion of hydrophilic R groups. In the unfolded state, they make lots of hydrogen bonds and dipolar interactions with water, keeping their enthalpy low and their entropy high (flexible unfolded conformation). The surrounding water is also able to benefit from those same enthalpic interactions and therefore doesn't need to awkwardly fit around the protein to maintain its own hydrogen bonding network (entropy high). Sequence determines structure, and the link between the two is energy.

Transcription

For transcription to occur, RNA polymerase must bind to the promoter. And for the RNA polymerase to bind, the two DNA strands must unwind. The promoter itself is a very AT-rich sequence. This is not a coincidence. The difference between AT and GC base pairings? The number of hydrogen bonds: 2 vs 3.

One hydrogen bond fewer makes it easier for the DNA strands to unwind. It requires less energy.

The same logic applies in PCR primer design, but in the other direction. A high GC (%) content on the 3' end of the primer ensures binding stability (3 hydrogen bonds formed release more energy than just 2!), meaning that it takes more energy for the primer to fall off, ensuring high PCR yield.

ATP hydrolysis

ATP is the product of respiration, the energy currency of the cell, and a great case study for understanding the difference between kinetics and thermodynamics.

ATP is kinetically stable but thermodynamically unstable. Let's look at why this is important. Thermodynamic instability simply means that the potential products are lower energy than the reactants. In other words, ATP hydrolysis releases energy. Which is exactly what we need it to do.

A simple case study to show this: if you leave ATP in a test tube with water for a day, it will end up as 100% ADP + Pi. Why is ATP hydrolysis thermodynamically favourable? Well, in simple terms, one molecule becomes two - a large increase in entropy. But also there is a change from ATP(4-) to ADP(3-) and Pi(2-), meaning increased charge-based interactions with water, so there is a loss in enthalpy.

However, this process takes place slowly. This is what we mean by ATP being kinetically stable: it has a relatively high activation energy for its hydrolysis to ADP and Pi, so the rate at which it takes place is very slow. But Biochemical reactions need to happen on the order of milliseconds.

So how is ATP actually used in cells then? ATP is only hydrolysed by enzymes, which lower the activation energy to unlock the thermodynamic potential of ATP. If this wasn't the case, and ATP was kinetically unstable, all of the ATP would hydrolyse all the time anywhere in the cell, losing all the energy to heat. All that respiration would be for nothing.

Instead, ATP hydrolysis takes place in the context of enzymes. Not only does this actually make the reaction happen on a biologically relevant timescale by lowering the activation energy, but it also ensures that the energy change is used to do work, rather than release heat. The substrate is phosphorylated using the free Pi released, producing a resulting conformational change which, for example, can impact signalling pathways.

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