Protein Archetype: The Chaperone

Yes, the cellular environment can be full of all kinds of snares for a young, not yet fully formed protein. New-come to the world, some proteins have to be chaperoned in their early life. If not, they might meet a bad end. And I’m talking a really bad end: like being whisked off and chopped up to bits. Thankfully, the strict, straight-laced chaperone proteins are present to help a new protein make something worthwhile out of itself.

Broadly Speaking:

In a cell, there are many proteins which function as “molecular chaperones.” Chaperone proteins are most commonly found in the cytoplasm as well as in the lumen, or inner portion, of the organelle called the endoplasmic reticulum. Chaperones in general have three main jobs to do. These jobs are: helping newly-made proteins get into the proper shape for their future careers, keeping proteins from clumping up together after a stressful event, and finally, refolding stressed-out proteins back into shape. The job description for molecular chaperones sounds more like that of a personal trainer than a Victorian-era spoil-sport!

But why do new proteins need help with their folding? Let’s start the answer to this question with a review on how proteins are made. As described previously, DNA (the master file) is transcribed into mRNA (copies of the file). Then the mRNA copies are translated into the building blocks of proteins called amino acids. Now, the amino acids are strung together by what is essentially a little machine made of proteins called The Ribosome (which is always depicted in a shape that looks a bit like Blinky from PacMan). The ribosome makes a linear chain of amino acids, but to become a fully functional protein, this chain needs to fold up into particular 3D shapes. Some proteins begin folding as they emerge from The Ribosome, but many proteins need a little help to reach their proper shape, or conformation. For those proteins which require aid, chaperone proteins are there to interact with the new protein while it is still being made and help the folding process.

As a new protein is being translated from mRNA by the Ribosome, it begins to fold up into certain 3D shapes, or conformations. Some proteins can fold into their proper, functional conformation without the help of a chaperone.

The proteins that need help contain stretches of certain amino acids which are “hydrophobic.” This term literally means that they are “water-fearing,” and it describes a physical property of these amino acids -- not wanting to mix or interact with water. As a more every day example of a substance that is hydrophobic, think of oil. If you put oil and water together, they separate from each other. In the same way, hydrophobic amino acids don’t like to be around water – and that’s an unfortunate predicament in a cell (which is about 70% water)! What’s a poor hydrophobic amino acid supposed to do? Does it face its fears? Nope, hydrophobic amino acids do what oil does in a similar situation; they huddle up, all stuck together or they find some other amino acids to hide behind.

Depiction of hydrophobic amino acids when exposed to the cytoplasm. Liberal artistic licence applied.

So, unsurprisingly then, hydrophobic amino acids are typically found in the interior part of a protein. In fact, this is true so much of the time that if hydrophobic amino acids are exposed on the outer part of a protein, the cell sees that as a signal of a protein which is not functioning properly. This is because proteins need to be folded correctly for their full function. Therefore, if these hydrophobic amino acids are on the outside, the protein must not be correctly folded. So the cell tries to either correct the problem, and sends in a chaperone, or it destroys the misfolded protein.

And so, even at the time of its birth – even as it is coming out of The Ribosome - the new protein could be in danger. And many new proteins do not escape that danger. It has been estimated that 25% of all proteins synthesized are destroyed because they did not fold correctly. You see, if the chaperone does not interact with the hydrophobic amino acid sequences, they are recognized by a different protein, tagged, and sent to another molecular machine called The Proteasome to be chopped up into pieces. Barbaric, right? But the cell has good reasons to be so strict, as I will now go over.

Some newly formed proteins require the help of a chaperone protein, shown in green, to correctly fold. Without help, these proteins are more likely to be degraded by the Proteasome.

So remember I said that hydrophobic amino acids like to stick together? This can cause big problems for the cell. If proteins are sticking together by their hydrophobic amino acids, not only is the protein not functioning properly, but it can stick to other hydrophobic areas on other proteins, leading to a messy cluster called an “aggregate.” In some cases, this misfolding and aggregation can cause or contribute to the development of serious disease. For instance, in the inherited diseases of sickle-cell anemia and alpha-1-antitrypsin deficiency, mutant proteins are able to escape the cell’s quality controls and form aggregates. These aggregates can severely damage cells and even cause cell death. In some cases, the protein aggregates are released from dead cells and accumulate around other cells in a tissue, leading to the damage of that tissue. Other diseases in which misfolded proteins are involved include Huntington’s, and Alzheimer’s, and Prion diseases. So, in summary, if cells cannot control the proper folding of their proteins, the result could eventually harm the organism. It makes sense to have strict measures in place to try to avoid harm.

Misfolded proteins with exposed hydrophobic amino acids can cause aggregations of proteins that can be harmful to the cell, surrounding tissue, and the organism itself. The presence of chaperone proteins helps to shield the cell from this harm.

Even after a protein is fully formed, it may need a chaperone again. This is because proteins are not 100% stable; they have regions of high and low stability. Under conditions which are stressful to cells, proteins can unfold a bit. This is called “denaturation” because proteins come out of their “natural,” folded state. Denatured proteins are usually still partially folded, but the loss of structure in one part of the protein can destabilize other parts. Stressful conditions which can denature proteins include high heat, high pH, and the addition of certain organic solvents/solutes. A description of how these conditions can denature proteins is found further down under the heading “Technically Speaking.”

So the second job of chaperone proteins is to help prevent protein aggregation during stress conditions. During a stressful event, the chaperones bind to the exposed hydrophobic amino acids and this interaction shields the other hydrophobic amino acids from a neighboring protein. In this way, chaperone proteins are a bit like their human namesakes – they keep apart things that would otherwise be attracted together in order to prevent future problems from arising. Or, it’s also a bit like enacting a curfew in troubled times in order to prevent frightened citizens from coming together into a store-front-destroying mob. This process of shielding unfolded proteins from each other is so important that during stressful conditions the cell decides to use its resources to create more chaperone proteins.

Stressful conditions can lead to fatal attractions. Luckily, the chaperone protein is there to block relationships that would be unwanted by the cell.

In the event that the cell survives the stress, it needs to be able to go on with business as usual. How can it do that when the stress has partially unfolded many of its proteins? Well, some proteins will snap right back into place after the stress is removed. Consider these particular proteins to be like Superman. The stress is like Kryptonite to them –the protein is functionally worthless until the Kryptonite is removed. As soon as the Kryptonite is gone though, bam, Superman is back in business, saving the world. This ability of a protein to be denatured and then “renatured” was first shown by a guy named Christian Anfinsen in the 1950s. It was an important experiment because it showed that the particular sequence of amino acids within a protein contains all the information needed for the proper folding of the protein.

Just as Superman cannot function with Kryptonite around, but then bounces back to his full strength when it is removed. So too can some small, stable proteins "renature" on their own after being denatured by stressful conditions. 

However, the process of snapping back into business after stress only works well for small and inherently stable proteins. For most proteins, some assistance is required. And that brings us to the final job of chaperone proteins, which is to refold proteins during and after stress. (Psst – I say the final job, but actually, different chaperones may have additional roles in the cell. More on that some other time, perhaps). After a hurricane, you try to salvage what you can. In the same way the cell would prefer to salvage as many of the proteins denatured by stress as possible, and so the chaperones get to work refolding the proteins. For this type of work, there is also a type of chaperone called a “chaperonin” which is barrel-like in its structure. Misfolded proteins go into this barrel, a “lid” is put on top, and the misfolded protein now has an isolation chamber of its very own which both prevents its aggregation with other proteins and provides a favorable environment in which to fold.

Like a pleasant trip to a mini-spa, the chaperonin allows a frazzled protein the peace and quiet it needs to pause, refresh, and prepare to face a new day.

You got to give these molecular chaperones credit. They tirelessly work to:

·         --preserve the wee newborns from destruction (ie properly fold new proteins)

·         --prevent mob formation for the city’s protection (ie prevent protein aggregation)

·         --run salvage operations after a hurricane hits (ie refold protein after stress)

Technically Speaking:

How do large proteins fold? –Its complicated and still being worked out, but there are several models. In the first, local secondary structures form first, like alpha helices and beta sheets, due to the ionic interaction of amino acids near one another on the linear protein chain. After this, assembly of more long-range interactions could come together to form super-secondary structures until the folding of the protein is complete.  In a different model, folding is caused by a spontaneous collapse of the amino acid chain into a compact state. This collapse into a compact state is caused by hydrophobic interactions. Most proteins probably use a mix of both models to fold. Also, protein folding may require other proteins besides chaperones, including protein disulfide isomerases and peptide prolyl cis trans isomerases.

How do stressful conditions denature proteins? - Most proteins can be denatured by heat, which has effects on the weak interactions in a protein (primarily hydrogen bonds). Extreme changes in pH can alter the net charge of the protein, causing electrostatic repulsion and the disruption of some hydrogen bonding. Organic solvents and detergents disrupt the hydrophobic interactions that make up the stable core of proteins.

How do chaperones recognize proteins that need their help? – Work is still being done to try to figure out how chaperones interact with their “client” proteins. However, while the presence of exposed hydrophobic surfaces is generally viewed the mechanism by which chaperones recognize and bind to their clients, other factors may also be at work. For instance, a small chaperone from E. Coli named “Spy” recognizes its clients through electrostatic interactions. Some data exists which suggests electrostatic forces may be important for other chaperones as well. Also, its interesting to note that certain chaperones will specifically interact with certain clients and not with others. The reasons for this specificity are beyond the scope of this review.

How do chaperones recognize that their job is done and they can release a newly folded client? - In some chaperones, ATP hydrolysis causes the client-binding site on the chaperone to change shape and this leads to the release of the client protein. In the case of the chaperone Spy, when the client’s hydrophobic amino acids are back into place, the lack of hydrophobic amino acids on the outside of the protein reduces the binding affinity of the client to Spy, resulting in the release of the client.

References:

Alberts, et al. Molecular Biology of the Cell. 5th Ed. New York, NY: Garland Sciences, 2008.

Nelson and Cox. Principles of Biochemistry. 5th Ed. New York, NY: W.H. Freeman and Company, 2008.

Koldewey, et al. “Forces driving chaperone action.” Cell. 2016. Jul 14;166(2):369-79. doi: 10.1016/j.cell.2016.05.054.