Protein folding game

Roll the dice!

Instructions

Decrease your Delta G by moving across the board, and become a more stable version of yourself!

Protein Folding Game

Oh No! Your polypeptide is at a local energy minimum- roll each turn until you get an odd number, then you can progress that turn thanks to a chaperone! This idea is detailed in the rules, but sometimes polypeptides can get stuck in a confirmation that is difficult to break out of because it has a significantly lower ΔG than similar conformations. This means that another party must supply energy to help that protein continue folding- a job of chaperones. These amazing and diverse proteins can aid in folding in a variety of ways- changing the local condition, mechanically shifting conformation, and stabilizing part of the polypeptide. Several example chaperone structures: Image from ‘How do Chaperones Bind (Partly) Unfolded Client Proteins? ’ I. Sučec , B. Bersch, P. Schanda from Frontiers in Molecular Bioscience, 2021 https://www.frontiersin.org/articles/10.3389/fmolb.2021.762005/full

Congratulations, your protein has achieved its most energetically stable tertiary structure! Roll a dice, if you get a four or higher, your protein has associated with other compatible proteins into a quaternary complex. If not, your protein remains independent. Multiple individual proteins can associate with each other into quaternary structures. These multisubunit structures are made of several individually folded tertiary structures held together by covalent and non-covalent interactions between them: ionic, disulfide, and hydrogen bonding, the hydrophobic effect, as well as Van der Waals interactions. Quaternary structures can be homotypic - made of the same type of repeating subunit like hemoglobin - or heterotypic - made of different subunits such as RNA polymerase.

If your protein has previously formed disulfide bonds, it will be recognized by other proteins for exportation from the cell. Roll a dice and move forward the appropriate amount of spaces. Proteins containing disulfide linkages are commonly exported from the cell. While the environment inside the cell is reductive, the oxidizing environment outside the cell helps to stabilize the disulfide bonds between oxidized cysteine residues.

Hip hip hooray! With the help of a PDI enzyme, your protein has formed the proper disulfide bonds. The most energetically favorable and stable three-dimensional configuration of a protein is called its native conformation. As the protein folds, cysteine amino acid residues come close to each other in 3D space. When oxidized, disulfide bonds form between them. These covalent bonds help to stabilize the tertiary structure of the protein. Disulfide linkages between the incorrect cysteine partners formed in the intermediate stages of the folding process may limit the ability of the protein to fold into its native structure, compromising the protein’s function. An enzyme called protein disulfide isomerase (PDI) catalyzes the formation and rearrangement of disulfide bonds. If a non-native disulfide bond forms during folding, PDI will reduce it and allow the protein to continue folding toward its native structure. Image from Adams, Benjamin M., et al. “Protein Quality Control in the Endoplasmic Reticulum.” The Protein Jurnal, vol. 38, no. 3, 2019, pp. 317–29.

Nice! Some of your amino acids formed into a beta barrel structure, that's electrostatically favorable! Advance three spaces.

Secondary (2°) structure is the local folding of small groups of amino acids, (usually around 12) into small, repeating subunits. There are two main subunits that make up 2° structure:

• Alpha helices - the polypeptide chain is organized into a corkscrew-like shape stabilized by hydrogen bonds between the carbonyl and amide groups
• Beta sheets - the polypeptide chain is organized into a flat line stabilized by hydrogen bonds between the main chain

Wahoo! Your protein has begun folding thanks to the hydrophobic effect.

Proteins start as unfolded(denatured) chains of amino acids. Some amino acids avoid water(hydrophobic). Unfortunately for them, there is a lot of water inside all cells of the body. The hydrophobic effect is the tendency of these amino acids to gather and “hide” from the water by moving to the interior of the protein. The remaining amino acids that love water(hydrophilic), move to the surface of the protein. This functions as the initial step of the protein becoming folded. Thermodynamics is also at play here, helping to drive this along. You see everything in the universe is trying to move towards its lowest energy state. What requires the least amount of energy? Well, disorder and unmitigated chaos. In science, we refer to this as entropy. In a nutshell, the more entropy (molecular randomness) you have, the less energy you need. Conversely, the more order you want, the more energy it will require. Just like making your house more orderly takes energy, so does ordering molecules. Water is in its lowest energy state, when it is free to move around and be disorganized. An unfolded protein, with its hydrophobic regions exposed, works against this by causing water to become more ordered. Water molecules bind to each other while avoiding the hydrophobic amino acids they are being pushed up against, this results in a cage-like structure. These are referred to as clathrate cages and can be seen in the illustration on the left. These caged water molecules cannot move freely and so exist in a higher energy state. When the hydrophobic amino acids move to the interior of the protein, this trapped water is liberated, free to rejoin the rest of the water in the surrounding environment. The net effect here is water going from a higher energy state to a lower one. This is thermodynamically favorable and so the process will naturally occur.

A chaperone has noticed your protein’s exposed hydrophobic regions! It has come to assist with folding!

There is a whole family of chaperones present in cells. Instead of following a one size fits all system, a variety of chaperones exist, each specific to their own respective protein type. Furthermore, there are chaperones specialized for individual steps in the folding process. While this chaperone cannot assist you with the entire folding process, it can aid you with some of your initial folding steps. Let’s see how. First check out the illustrations on your left, then read the text below.

As you can see in the illustration titled GroEL/GroES Complex, a chaperone consists of three rings. Each of these rings is made of 7 proteins, 21 in all. There are two identical GroEL rings stacked on one another, taking on a barrel shape and one smaller GroES ring that functions as a cap. Your protein’s hydrophobic regions are drawn to the chaperone’s hydrophobic stripe, and it enters a hollow compartment within the “barrel”, then the GroES cap covers the top like a lid. At this point your protein is isolated from the busy cellular environment outside. The protein then moves to a folding cavity within the chaperone. It is here your protein can accomplish aspects of its folding process without the risk of misfolding or becoming clumped together with other unfolded proteins(aggregation). Not only do chaperones act as a shield for your protein, they also directly aid in folding. They do so in a regulated way, like a builder using blueprints. Chaperones follow a specific folding pathway towards the lowest energy state, also known as the native state. When the chaperone finishes with the parts of folding it can help with, it releases the protein.

Now that the chaperone has released your protein, go ahead and take a trip down the slide!

Instructions

Hopefully, many of you have played snakes and ladders before. However, this time the goal is to descend to the lower ΔG (Delta G) native conformation. Be sure to read each event that you land on! Even before and after interacting with ladders and snakes! Hover over the icon to read the event tooltip, click on the icon for a more in depth explanation and rules in bold. !If you and another player’s polypeptide occupy the same space, you must restart! Protein aggregates can form for a variety of reasons, but they can have intermolecular interactions with other polypeptides (often of the same kind) strong enough that they can become stuck in groups. This can be prevented by chaperones- a diverse group of proteins that help polypeptides fold. Good luck!

Each player starts with a token on the starting square on the top left. Players take turns rolling a single die to move their token by the number of squares indicated by the die roll, making sure to interact with every icon they land on. Tokens follow a fixed route marked on the gameboard which usually follows a track from the top to the bottom of the playing area, passing once through every square. If, on completion of a move, a player's token lands on the lower-numbered end of a "chute", the player moves the token up to the chute's lower square. If the player lands on the higher-numbered square of a ladder, the token must be moved down to the ladder's higher square. If a 6 is rolled the player, after moving, immediately rolls again for another turn; otherwise play passes to the next player in turn. The player who is first to bring their token to the last square of the track is the winner.

Why do we care? Aside from protein folding being required for them to function- and those functions being required in order for you to be alive- many diseases occur as a result of misfolded and/or aggregating proteins. Several neurodegenerative diseases are caused by protein folding being changed by a different primary sequence as in Huntington’s disease or aggregate formation as in Alzheimer’s. Misfolded proteins can take on a malignant nature all their own in the form of prions where these misfolded proteins can cause other correctly-folded proteins to become misfolded and cause infectious ailments such as Mad Cow Disease and Chronic Wasting Disease. But what on earth is ΔG? This term may be unfamiliar or bring feelings of intense confusion and fear- but fear not. ΔG, short for Gibb’s free energy, is a concept relating to spontaneity and stability- think of it as how at- ease the universe is with a certain arrangement. ΔG=ΔH-TΔS is the state function- dependent upon enthalpy (ΔH) and entropy (ΔS) as well as the temperature (T). Delta G being negative in this context means that the conformation in question, with its own enthalpic and entropic interactions with itself and the surroundings, is stable - the more negative the more stable.

CHUTES

LADDERS

If the player lands on the higher square of a "chute", the token must be moved down to the snake's lower square, essentially going forwards

If the player's token lands on the lower end of a "ladder", the player moves the token up to the ladder's higher square, essentially going backwards

Oh No! Your polypeptide is at a local energy minimum- roll each turn until you get an odd number, then you can progress that turn thanks to a chaperone! This idea is detailed in the rules, but sometimes polypeptides can get stuck in a confirmation that is difficult to break out of because it has a significantly lower ΔG than similar conformations. This means that another party must supply energy to help that protein continue folding- a job of chaperones. These amazing and diverse proteins can aid in folding in a variety of ways- changing the local condition, mechanically shifting conformation, and stabilizing part of the polypeptide. Several example chaperone structures: Image from ‘How do Chaperones Bind (Partly) Unfolded Client Proteins? ’ I. Sučec , B. Bersch, P. Schanda from Frontiers in Molecular Bioscience, 2021 https://www.frontiersin.org/articles/10.3389/fmolb.2021.762005/full

Oh No! Your polypeptide is at a local energy minimum- roll each turn until you get an odd number, then you can progress that turn thanks to a chaperone! This idea is detailed in the rules, but sometimes polypeptides can get stuck in a confirmation that is difficult to break out of because it has a significantly lower ΔG than similar conformations. This means that another party must supply energy to help that protein continue folding- a job of chaperones. These amazing and diverse proteins can aid in folding in a variety of ways- changing the local condition, mechanically shifting conformation, and stabilizing part of the polypeptide. Several example chaperone structures: Image from ‘How do Chaperones Bind (Partly) Unfolded Client Proteins? ’ I. Sučec , B. Bersch, P. Schanda from Frontiers in Molecular Bioscience, 2021 https://www.frontiersin.org/articles/10.3389/fmolb.2021.762005/full

This game is heavily based upon the idea that polypeptide folding occurs in a landscape representing complex internal and external interactions as it is folded into its native conformation (the conformation required for it to do its job). Levinthal’s paradox was an earlier theory that protein folding was a completely random process, but if that were true it would take more time than the universe has existed just for some of them to fold! But in reality these polypeptides are folding all at once in a variety of ways- This gives us our funky folding funnel, where ΔG is decreasing and as it does so the number of conformations decrease as we approach the native fold. But watch out! This landscape is complex, and some conformations that the polypeptides find themselves in may be cozy, but not the native conformation- this is one of the reasons chaperones are so important, as they can catalyze movement out of these local energy minima.

You're currently an unfolded peptide! Progress to fold and decrease your ΔG.

As you might already know, decreasing your Delta G means to become more stable. When you’re just a string of amino acids floating in space, you aren’t very stable and there are forces already working to pull yourself together. As you progress along the board, your form becomes more stable and the forces that pull and push you into a protein become more prevalent, decreasing the Delta G of your protein folding. Some events might propel you further down the board, which are our chutes, and some events might send you back into a more unstable state, which are our ladders. Sometimes your protein misfolds and you get stuck in a weirdly stable, but unusable state and you need to expend some energy to get out. As you go down the board you’ll see many different real life examples of this, and understand the mechanisms that both increase or decrease your Delta G. Image from ‘On a generalized Levinthal's paradox: The role of long- and short range interactions in complex bio-molecular reactions, including protein and DNA folding’ A. Melkikh, D. Meijer from Progress in Biophysics and Molecular Biology Vol 132, 2018

Your protein is being examined for a targeting signal that determines its localization within or outside the cell. Roll a dice! If the number is even, your protein is recognized for transport to the endoplasmic reticulum, take an extra turn! If the number is odd, your protein will remain in the cytosol and continue folding. Proteins will either remain in the cytosol or be transported to the endoplasmic reticulum (ER) as they are translated. Some proteins have an amino acid sequence known as a signal peptide that functions like an address label, tagging a protein bound for a specific organelle such as the Golgi apparatus, lysosome, the plasma membrane, or the exterior of the cell. Proteins with these signals, often a series of hydrophobic residues near the N-terminus of the protein, will be recognized by other proteins in the cell which will help transport it to the right destination.

Your protein will need help from a PDI before it can continue folding! Roll a dice each turn until you get a three or lower to attract a wandering PDI. To avoid the potential risks of misfolding and aggregation posed by the formation of improper disulfide linkages, an enzyme called protein disulfide isomerase (PDI) catalyzes the formation and rearrangement of disulfide bonds. PDI will reduce incorrect disulfide linkages, allowing the protein to continue folding toward its most stable native structure. Image from Adams, Benjamin M., et al. “Protein Quality Control in the Endoplasmic Reticulum.” The Protein Journal, vol. 38, no. 3, 2019, pp. 317–29.

Oh No! Cellular stress! Better get some heat shock proteins.

It’s important to remember that what happens to your body, happens to your cells. When you sprint, lactic acid builds up in cells, which affects pH levels. If you summit Mt. Everest, your cells will have less oxygen in them. If you sit in a sauna, your cells heat up. We explained earlier how a protein works towards its lowest energy state, its native state. It’s important to note that the “lowest” energy state is relative to the current environment of the cell. If you raise the temperature, the old native state can no longer be maintained. The protein partially unfolds because it can no longer stay at such a low energy conformation. The protein will try to refold but now only has the “current” lowest energy state to work towards. Which might be relatively higher than the old native state. This is a big deal, only proteins in the native state determined by normal cellular conditions function and perform necessary tasks. Proteins and the molecules they interact with are often related to a lock and key. Their compatibility is highly specific. If you refold the protein into some other shape, it would be like trying to use a different key in a lock. Thankfully, the cell is adapted to address this very scenario. When cellular stress is recognized, the cell immediately begins producing what are called heat shock proteins. These proteins are a type of chaperone, like the ones we introduced earlier. These chaperones are adapted to refold proteins denatured by cellular stress. Additionally, these chaperones attach to proteins, temporarily stabilizing them. Thus, preventing further denaturation from occurring while the cell remains in a stressed state. Cells are regularly being stressed for one reason or another, so it is a good thing we have heat shock proteins!

Roll the dice. If you get 1-3, then your cell has returned to its normal temperature and the chaperone will assist you in refolding. If you roll 4-6, you lose one turn, your cell still needs to cool off!

Uh oh! Your protein has formed one or more improper disulfide bonds. The most energetically favorable and stable three-dimensional configuration of a protein is called its native conformation. As the protein folds, cysteine amino acid residues come close to each other in 3D space and become oxidized, allowing disulfide bonds to form between them. These covalent bonds help to stabilize the tertiary structure of the protein. Improper disulfide bonds form when cysteine residues form disulfide linkages with the incorrect partner, which can result in a different 3D folded structure. The formation of disulfide bridges limits the total number of potential conformations the protein can fold into. Improper disulfide linkages formed in the intermediate stages of the folding process may limit the ability of the protein to fold into its native structure, which compromises the protein’s function. These improper linkages may also expose hydrophobic regions of the protein to the surface, which can result in misfolding and potentially aggregation.

Hello protein, your human thought it was a great idea to attempt a marathon without any training!

The muscle cells(myocytes) of your human’s legs are accumulating copious amounts of lactic acid. This has lowered the pH of these cells and caused cellular stress. Your protein has partially denatured and needs the help of heat shock proteins to refold properly.

Roll the dice, if you get an even number, then your human has given up and stopped running. The cell pH level will return to normal, and your protein can refold with the help of heat shock proteins. If you roll an odd number, then your human has decided to keep running. Heat shock proteins bind to your partially denatured protein and prevent further denaturation while the cell stays in a stressed state. Unfortunately, this means you must take the ladder!