Monday, February 25, 2013

The human machine: probing the mechanics

The previous post in this series can be found here.

This week, inspired by Shaun's most recent post covering exciting new results in cosmology, I have decided to also take a quick look at one of the fascinating recent findings of molecular biology. I hope to give some insight into how this work is done, and why it is not only intellectually interesting, but also potentially practically useful. 

What do we know?

Those of you who have been following this series for a while might remember a post that I wrote last year (biological batteries and motors) where I discuss how energy is converted from myriad chemical forms in your food into the single energy currency of the cell, ATP. The system by which this is achieved is quite beautiful, chemical energy is converted into an electrical current within the mitochondria of your cells, which is in turn converted into a current of protons. This proton current drives a motor (ATP synthase) that churns out ATP, thereby converting it back into chemical energy. I'm not going to go into the whole process again here, but if you'd like a quick refresher then just hop back to my older post here, go on - you know you want to! I don't mind waiting.

So, a key player in this whole process is the so-called respiratory complex I (or NADH dehydrogenase), which is the first link in the chain that converts electrical current into proton current. Complex I takes electrons from a molecule known as NADH, which is produced from energy in your food by a range of complex metabolic chemical reactions. It moves the electrons that it takes from NADH and sticks them onto a molecule called ubiquinone, which then moves on to the next stage in the process: the perhaps confusingly named complex III.

Complex I is not just a relay for electrons, though. In fact it also directly moves protons from one side of the mitochondrial membrane to the other, thereby contributing to the aforementioned proton current that drives ATP synthase. For every electron that it transfers from NADH to ubiquinone, four protons get moved across the membrane. Until recently we knew little more than this because we didn't really know what complex I looked like. Molecular biologists had been able to purify it out from our cells and determine that it is pretty massive (containing 44 individual proteins arranged together) and identify a couple of candidate proteins in that mess that might be responsible for some of the complex's functions, but we didn't know how they all fitted together and so how they might work on a large scale. This is important because mutations in complex I are the most common cause of human neurodegenerative diseases. This is because a mutated complex I can go a bit wrong and start sticking electrons where it isn't meant to - most significantly onto oxygen-containing molecules to generate reactive oxygen species that cause damage to mitochondrial DNA and so impair neuronal function in particular. Parkinson's disease and Leigh's disease are two of the more common disorders associated with complex I, but there is also evidence that the speed of ageing is also linked to the activity of your complex I assemblies. It is, needless to say, quite an important protein.

Recent developments

As you may have already guessed from the overall tone - there has recently been some light shed on the mystery that is complex I. Last week, Nature published a study by Leonid Sazanov's research group in Cambridge in which they uncover the full structure of complex I for the first time. Bits of the structure had been known before, but this is the first time that we've had the whole thing revealed before us!

Sazanov's group have achieved this by using a technique commonly used in structural biology - X ray crystallography. I touched briefly on crystallography in my post looking at G protein-coupled receptors in case the term seems vaguely familiar. The problem that arises when looking at very small things is that light is actually too big to use as a tool to see things. By "too big", what I mean is that the wavelengths of what we would generally call 'light' (i.e. the visible and near-visible spectrum) are actually larger than the fine details of the thing you want to look at, so there is an inherent maximum resolution that can be achieved. In order to go into finer detail we need to use electromagnetic radiation with a much higher energy and so shorter wavelength - X-rays.

When you fire a beam of X-rays at a protein, some of the X-rays interact with the electrons in the protein's constituent atoms. These interacting X-rays are then scattered out in various directions and so if you detect them then you can use some clever tricks to work out how the atoms in the protein are arranged. However, the vast, vast majority of X-rays will not interact with the electrons in your protein and the scattering will be pretty damn weak. In order to bulk up the signal you need to get many copies of your protein (millions of millions of copies) and arrange them into an ordered fashion so that they all scatter in the same way. To do this you have a grow your purified protein into a crystal where each one is identical to the next and in exactly the same orientation. This is the tricky bit. Biochemists have been making protein crystals for over half a century with great success, but the bigger and more complicated your protein is then the harder it is to crystallise. Moreover, if you protein usually sits in a membrane then it makes it even more difficult to force into a crystal. Complex I ticks both these boxes and so making a successful crystal is nothing short of a scientific miracle! They don't say in their paper, but I imagine that Sazanov and colleagues have been trying to get this crystal for a LONG time! It was, however, undoubtedly worth it. The structure is quite beautiful.

The structure of respiratory complex I - taken from Baradaran et al, Nature, 2013.


The multi-coloured structures in the figure above represent the individual proteins involved the complex, with different peptide structures represented as different shapes (ribbons, loops etc). What is immediately apparent is that there is a large region that is embedded in the membrane (the flat bit at the bottom) and a separate segment that extends upwards and away from this membranous section (the bit at the top right). Analysis of the amino acids present at different regions reveal that the top-right protrusion is the binding site for NADH and ubiquinone, and so is where electron transfer must occur. Conversely, the membrane-embedded regions are where the protons get pumped across the membrane, so the two processes must be coupled in some way.

This structure allows us to suggest mechanisms in which the two might be coupled as it identifies 4 distinct channels through which protons can cross the membrane. As I mentioned earlier - the transfer of one electron from NADH to ubiquinone results in the movement of 4 protons across the membrane, so the fact that there are 4 proton channels seems to fit neatly with the idea of one proton moving through each channel.

Three of the four proton channels identified by Baradaran et al. The paths that protons take through the protein are shown as blue arrows.
Looking at both the electron-transporting and proton-transporting sections has allowed the creation of a model of just how the two transfer processes are linked. To start with, the proton channels are open at the end facing into the cytoplasm (the side of the membrane from which protons will be removed) and closed to the periplasm (the side to which they will be released). The transfer of an electron from NADH to ubiquinone causes a wave of shape changes that start in the non-membrane embedded region and then cascade throughout the whole protein complex. This causes the proton channels to switch so that they are now closed to the cytoplasm and open to the periplasm; free to release their protons. Thereby, 4 protons are moved over the membrane for every electron transferred.

Electron transfer coupled to proton transfer. (NB. H+ is a proton in this representation)

The complexity of this whole system is quite astounding, doubly so when you consider that every complex I in your body is doing this thousands of times every second, and that there are billions of billions of these complexes in your whole body. What's more, now that we know how it works, we can start to make sense of how it can go wrong in neurodegenerative disorders and so potentially one day come up with effective treatments that benefit millions of people.

The story of complex I is an excellent example of just how unbelievably well engineered we are as biological machines, yet also how ingenious we can be and have been in our efforts to understand our own engineering. Moreover, it's a demonstration of how research is always progressing in the background, largely ignored by wider society but still delving deeper and uncovering more about what and who we are.

The next post in this series can be found here.


Baradaran, R., Berrisford, J., Minhas, G., & Sazanov, L. (2013). Crystal structure of the entire respiratory complex I Nature, 494 (7438), 443-448 DOI: 10.1038/nature11871

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