Blobology and proteins' little helpers

Molecular chaperones are a set of families of proteins whose function is to help other proteins to fold, unfold and generally keep them out of mischief. When something goes wrong in protein folding, such as the unfortunate tendency to form aggregates, molecular chaperones have a distinctly important role to play.

One of the most studied of the molecular chaperones is the chaperonin family, a group of large, double-ringed proteins that form structures in which other proteins fold. The most commonly studied member is GroEL, an E.coli protein needed for bacteriophage growth. It acts in concert with a co-helper, GroES, to form a unique protein container in which polypeptide chains are safe to begin to fold free from contact with other proteins and the dangers of aggregation.

Professor Helen Saibil has been at the forefront of the study of GroEL and other molecular chaperones for the last two decades. A physicist by training who became fascinated with how living things worked, she has worked at Birkbeck College London for almost 20 years, arriving there after stints in Paris and Oxford, following her PhD at King's College London under the supervision of Maurice Wilkins.

Saibil admits that she didn't realise it was that Maurice Wilkins when she first applied to King's, but they quickly became firm friends, remaining so until his death in 2004. "He was working on photoreceptor membranes and I was working on the physics of how things worked in biology," Saibil says. "He was a lovely man, extremely nice. I ended up staying a long time at King's learning how to do all sorts of things."

Now at the School of Crystallography at Birkbeck and director of its Bloomsbury Centre for Structural Biology, Saibil didn't start off as an x-ray crystallographer. "I never did x-ray crystallography exactly - I started off in physics and then I moved into biophysics. I was interested in how things worked and I still am. Biophysics seemed to be the way to tackle the molecules and the machinery of living things, and so it was the topic that drew me in.

"Vision in particular was an area that attracted a lot of people from a physics background as it was a mixture of optics and biology and it seemed that it was all electrical - the way light went into the eye and activated currents. I was drawn to the subject and in the lab (at King's) they happened to be using x-ray scattering to study photoreceptor membranes. I ended up doing neutron scattering on photoreceptor membranes, looking at the distribution of a protein in the membrane. We were trying to understand how light is turned into a visual signal."

Then electron microscopy came along. By this time Saibil was at Birkbeck, where a colleague had grown the first crystals of GroEL. He encouraged her to have a look at them on the EM and she was entranced. "I could see from the images that it must be possible to find out a lot from EM but I didn't know how at that time. It looked like something that I could really get my teeth into. I've been working on it ever since."

Lid on the box

GroEL is part of the heat shock protein (Hsp) 60 system, responsible for assisting protein folding. It is a hugely abundant protein in E.coli, often overexpressed when the cell is subject to heat stress, and is very easy to make - hence it's interest to researchers.

"It is also a very conserved protein so it's pretty much the same in humans and other species," Saibil says.

"GroEL is an interesting one because it is ubiquitous and it really helps proteins to fold. It doesn't just stop them aggregating but seems to actively help proteins to fold in ways which we still don't quite understand.

"Not only that but it makes a container inside which another protein goes and folds. So it's a huge box of protein for other proteins to fold in - it's a piece of machinery that is really quite fascinating."

GroEL works closely with GroES, which makes a lid to cover the open cavity of GroEL's container. "GroEL goes through huge conformational changes, including the binding of the Gro ES lid to make a closed box for protein folding," Saibil says.

"One non-folded protein ends up inside this box and by some mysterious means it gets caught in the hydrophobic binding site. Then when the box forms, all of the pieces of the box twist around so much that the hydrophobic sites get pulled off the non-native protein, and it ends up trapped inside a hydrophilic chamber where there is nothing it can stick to.

"It can't aggregate because it is all by itself and it is confined and can't stretch out in all directions because it is inside a closed container, which is in some cases only a bit bigger than the protein itself. And GroEL manages to make things fold that way - it's quite amazing."

The role of the molecular chaperones in preventing aggregation is hugely important. It was long thought that all the protein needed to fold up into its final three-dimensional shape was the genetic instruction manual. Now, it is known that proteins can get stuck along the way. When proteins start to fold, their hydrophobic parts are sometimes transiently exposed in the process, with those parts showing a liking to stick together and make aggregates.

Aggregates, for reasons still not fully understood, are extremely toxic to the cell and are the basis of many diseases like Alzheimer's and Creutzfeldt-Jakob. "Protein aggregation is something which is extremely damaging and which the cell has invested a huge amount of quality control to stop happening," Saibil says.

"The chaperones are quite a diverse group but one of the most common features is that they prevent aggregates from forming. They generally do that by binding the hydrophobic species and non-native species that are trying to aggregate or ones that are prone to aggregate."

---PB--- Blob-ology

The advent of electron microscopy - in particular single particle cryoEM - has revolutionised our understanding of these fascinating conformational changes in the molecular chaperones and other proteins. With EM it has been possible to trace the pathway of these changes, initiated by the binding and hydrolysis of ATP, which then drives the allosteric cycle observed in GroEL.

Before the development of EM, scientists worked in the field of 'blobology' - "everything just looked like blobs", Saibil says. "But even at the blob level, GroEL looked quite interesting.

"Ten years ago we made a series of little movies that we still show of the conformational changes in the GroEL system at 25 angstrom resolution. You could just see the shapes of proteins in their separated domains or subunits - you could see the symmetry. But nowadays we are getting resolutions of seven or eight angstrom and you can see the alpha helical densities. We are able to see the internal features of the protein in quite a lot of detail.

"People are developing this for other things like viruses. With GroEL now, people are using it as a test object and getting very good resolution on it by electron microscopy. So it's now possible to look at protein complexes in quite a lot of detail by these methods. And that's all happened at the same time as we have been working on the GroEL story."

In part due to her interest in the conformational changes observed in the molecular chaperones, Saibil has also ventured into the world of bacterial pore-forming toxins, a group that includes the anthrax toxin. Saibil has been studying pneumolysin, a pore-forming toxin secreted by Streptococcus pneumoniae that is part of the cholesterol binding family of bacterial toxins. They are characterised by the formation of large rings on the surface of cholesterol-containing membranes through which the toxin enters the cell or allows its contents to leak out.

"It is the same idea as the big conformational changes in protein complexes," she says. "Pore-forming toxins have to go through a huge change because they start as small, individual, soluble proteins - monomers - and then they assemble into huge rings that jump into membranes. Proteins are not supposed to do that - they are supposed to be soluble or membrane proteins. Normally they stay one or the other. But pore-forming toxins are fascinating for the reason that they go through huge changes and it is mysterious as to how a protein can do both of those things."

Protein misfolding

As well as looking at how molecular chaperones assist protein folding, there is the obverse - what happens when it all goes horribly wrong and protein aggregates form into amyloid fibrils. While it is not known exactly how they do it, amyloid fibrils are thought to be the toxic agents responsible for nerve cell death and loss of brain function in a number of diseases, including Alzheimer's and CJD.

Saibil often wishes she hadn't gone into this area as misfolded or denatured proteins are extremely difficult to work with. "Misfolded proteins look pretty disgusting and you can't recognise much," she says. "But there is a final destination point for a lot of these aggregates, which is amyloid fibrils."

From a structural perspective, amyloids are interesting because the proteins, whatever their starting structure was, are converted into a beta sheet structure. When proteins fold they make two regular, secondary structures. The polypeptide chain either curls into a spring shape (an alpha helix) or what Saibil describes as a Venetian blind, in which each slat is hooked onto the next, known as the beta sheet structure.

The spring-shaped type has hydrogen bonds along its length, folding it into a coil, and the beta sheet has them folding into a flat shape. "For some reason that secondary structure ends up in these fibrils and it doesn't matter what the starting structure of the protein was - all of them seem to end up in what is called a cross-beta fold," she says. "The beta sheets are perpendicular to the long axis of the fibre. So it is like very long ribbons of Venetian blinds.

"We have been looking at the structure of these fibrils, which is quite difficult. We haven't yet got very far but we hope it will eventually help us understand this mysterious conversion of the protein that has become denatured or messed up in some way. If we can understand the conformation of the fibril better we might be able to work backwards to see what the conversion is."

Saibil last visited Australia a decade ago, when she visited for the 23rd Lorne Protein conference. For the 33rd conference, she is an invited speaker. If she has time, she will discuss her research into the heat shock protein 104 (hsp104), a chaperone from the large hsp100 family found in bacteria, plants and yeast that seems to reverse aggregation.

The hsp100 subset is part of the large AAA protein family, ATPases that are involved in a number of different unwinding and unfolding helicases. "We've got a not particularly high resolution structure of hsp104, which I'll show if I have time," she says.

What she will talk about is the fascinating molecular chaperone story as well as more recent research, particularly the results of studies of what a non-native protein actually looks like when it is stuck inside the chaperonin. And she has pictures.

"We have some new results on a very large substrate on a bacteriophage co-protein," she says. "It's at the upper end of the substrate side that will fit into the chaperone box. We have a map now of the structure of the chaperonin container bulging at the seams, with a large protein stuck inside it."