Protein Folding & Quality Control

Summary

Cells constantly synthesize proteins, some of which will become misfolded. Survival depends on a balance of synthesis, proper folding, and removal of misfolded proteins. This balance is sometimes called proteostasis, for homeostasis of the proteome.

This article expands on the protein structure concepts introduced in Campbell, Chapter 5 (Macromolecules) and in the Biological Chemistry Overview page in my Canvas site.

Protein quality control video:

I made a lecture video on this topic. It's over 20 minutese long, so I thought you'd benefit from a written version for study and review. This page covers almost everything in the video. To do well on the quiz, you’ll need to study this page; the video gives a slightly different presentation of the points on this page (you could skip the video if you prefer, but don’t skip the reading and the review section below!).

Objectives

By the time you complete this unit, you should be able to explain the core concepts on this page:

  • How proteins fold in cells.
  • How protein folding can go wrong, and how this can affect the cell.
  • How cells use protein quality control mechanisms to reduce misfolding and aggregation.

For more detailed objectives, see the Review section at the bottom of this page. These concepts will be covered on the Proteins & Metabolism quiz on Canvas, and on Midterm 1.

Background: 

Before you start this page, it’s best if you read Campbell, Chapter 5 and the Biological Chemistry study guide, and complete the Biological Chemistry Quiz in Canvas. You should be familiar with amino acids and polypeptides.

Introduction

Proteins: precisely controlled, but flexible

Protein structure is complex. The range of possible protein structures is immense, partly because there are a lot of amino acid monomers to choose from, and those monomers are chemically very different from one another. That isn't true of DNA (which uses only four different nucleotide monomers) or polysaccharides (which are usually repetitive chains of only one kind of sugar monomer). The complexity of proteins allows them to be fine tuned to recognize specific molecules and carry out specific functions. That's why most of the specific catalytic and regulatory jobs in cells are done by proteins, and not by some other kind of molecule. Each protein’s function is determined by its precisely controlled shape and the chemical properties of its amino acid side chains.

PFKOn the other hand, protein structure isn’t rigidly fixed. Each protein’s overall conformation (shape) is influenced by numerous weak bonds and interactions, making proteins capable of changing their shapes in response to interactions with other molecules.

This picture shows a ribbon diagram of a protein called phosphofructokinase (PFK). It's an enzyme that catalyzes a key step in the process of glycolysis, which you'll learn more about when we get to cellular respiration. This enzyme latches on to a specific kind of sugar and modifies it in a precisely controlled way (as you'll see in the cellular respiration unit).

The ability to recognize a specific molecular partner is determined by the shape of the enzyme. In addition, PFK's shape can be modified, switching the enzyme's shape from an active conformation (in which it catalyzes a chemical reaction) to an inactive conformation (in which it doesn't). All your cells have numerous copies of PFK, and each of these proteins is continually being switched back and forth between its active and inactive forms, fine-tuning your body's ability to process sugars and use energy.

The complexity and flexibility of proteins is both an opportunity for the evolution of molecules that do very specific jobs, and a potential problem. Protein function is determined by protein structure, but a polypeptide can potentially get folded into the wrong structure, causing problems for the cell.

For this reason, cells need a quality control system for proteins: a set of mechanisms to deal with misfolded proteins. In recent years, researchers have increasingly found that many seemingly unrelated diseases are connected to problems of protein misfolding and the failure of quality control systems.

Four levels of structure

Four levels of protein structure

This diagram summarizes the four levels of protein structure. Campbell (Chapter 5) also has a good diagram (Exploring Levels of Protein Structure) showing the levels of structure and the specific kinds of bonds and interactions that are involved. You should look closely at that diagram, but here's a summary:

  1. Primary structure: The sequence of amino acids. The amino acids are joined by covalent peptide bonds, forming a polypeptide. The primary structure is determined by the sequence of nucleotides in the messenger RNA, so it can be predicted from DNA or RNA sequences. The term primary structure specifies only the order; it says nothing about shape.
  2. Secondary structure: Repetitively folded segments of the polypeptide, held together by hydrogen bonds to form either a helix or a sheet. Most proteins have multiple secondary structures.
  3. Tertiary structure: The overall 3-dimensional shape of a folded polypeptide, influenced by primary and secondary structures and controlled by numerous interactions within the polypeptide and with other molecules. ("Tertiary" just means third.)
  4. Quaternary structure: More than one polypeptide joined together to create a stable functional unit. Many proteins must be in quaternary structures to do their jobs. Quaternary structures can be maintained by weak interactions or strong covalent bonds.

Proteins can be described in terms of four levels of structure. However, this kind of description doesn’t necessarily say how a protein reaches its final tertiary and quaternary structure.

Protein folding

Protein folding

Each protein starts out as an unfolded polypeptide: a chain of amino acids produced by a ribosome in the process of translation.

To reach its final functional form (called the native conformation), the polypeptide must go through a controlled process called protein folding. The process of folding is influenced both by the polypeptide itself and by its cellular environment.

Overall, the primary structure is created by stable covalent bonds, but the other levels of structure are dominated by numerous less-stable interactions.

Hydrophilic and hydrophobic

cytosolic protein folding

Protein folding begins with hydrophilic and hydrophobic interactions. Polypeptides usually have some amino acids with hydrophilic side chains, and some amino acids with hydrophobic side chains (see Campbell, Ch. 5 for some examples of these).

Polypeptides in the cytosol (the watery interior) of a cell are surrounded by water molecules. The water molecules hydrogen bond with the hydrophilic side chains of some amino acids, while pushing away other amino acids with hydrophobic side chains. The end result is that the polypeptide folds toward a tertiary structure in which the hydrophobic regions are pulled together toward the interior of the protein, while the hydrophilic regions are pulled toward the exterior, where they interact with water.

On the other hand, some polypeptides are destined to become membrane proteins. These polypeptides contain many amino acids with hydrophobic side chains, and the hydrophobic groups get inserted into a membrane, where they interact with the hydrophobic interior of the membrane. 

An energy landscape

Energy landscape

Unfolded polypeptides aren't very stable; they're likely to fold onto themselves or stick to other molecules. Each time a polypeptide forms an interaction such as a hydrogen bond or hydrophobic interaction, it becomes more stable. In other words, as a protein folds, it moves toward a lower-energy, higher-entropy (more stable) state.

However, there may be more than one way for a protein to fold. Before reaching the native state, a polypeptide may get stuck in a stable, partially folded state, like one of the small valleys shown on this energy landscape diagram.

Partially folded, or misfolded, proteins can have disastrous consequences for cells.

Aggregation

Protein quality control

Once a protein has folded on itself and achieved its native conformation, it can interact in a controlled way with other proteins. These interactions are important, because proteins rarely act alone.

However, it's also possible for a partially folded or misfolded polypeptides to stick to each other. Misfolded proteins tend to have exposed hydrophobic regions, which are repelled by water and stick to other hydrophobic structures. Hydrophobic interactions can make a group of partially folded proteins stick together into a mass, called an aggregate.

Protein aggregates can be disordered blobs, or they can form ordered structures such as the amyloid fibrils shown in this diagram. The accumulation of amyloid fibrils in the brain is one of the hallmarks of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In addition, many other diseases, such as Sickle Cell disease, Mad Cow disease or Variant -Jakob disease, are caused by protein aggregation. These diseases have distinct causes and symptoms, but all involve the aggregation of proteins due to exposed hydrophobic regions.

Protein aggregation can be a disaster for a cell, because the aggregated misfolded proteins don't function properly, and because they can accumulate until they overwhelm the cell's normal processes.

Quality control

QC chaperone bigCells need to make sure proteins get folded properly and don't form aggregates. In other words, cells need protein quality control systems.

This diagram shows a misfolded protein with several possible fates. Ideally the misfolded protein becomes a properly folded native protein. Alternately, it could turn into several kinds of dangerous aggregate. Chaperone proteins play critical roles in the quality control system, pushing proteins toward their native conformations, or helping destroy those that cannot be properly folded. In this diagram, the green lines represent quality control pathways promoted by chaperones, while the red lines represent "off-pathway" misfolding and aggregation.

A safe environment

Bacterial chaperoneChaperones can prevent unfolded polypeptides from forming aggregates. One of the ways they do this is by providing a safe environment for folding, away from the potential influence of other unfolded proteins. "Safe" in this case means an environment in which there's nothing for the hydrophobic regions of the polypeptide to stick to, except other parts of the same polypeptide. The chaperone promotes self-folding rather than aggregation.

This picture shows a well-known bacterial chaperone (called GroEL-GroES). It acts like a container, taking in unfolded polypeptides and releasing then when they're folded. (See Chaperones listed in the references section below, for more on how this process works).

Ubiquitin labels proteins for destruction

UbiquitinationProper folding is one side of protein quality control; the other side is the destruction of proteins that can't be folded properly, or simply aren't needed in the cell. Many of these proteins are destroyed by structures called proteasomes.

Proteins that are destined to be destroyed in proteasomes are first labeled with another small protein called ubiquitin, in a process called ubiquitination. A long chain of ubiquitins is attached to the target protein, causing it to be taken to the proteasome and destroyed.

Ubiquitination of proteins can have many different effects in cells, depending on how many ubiquitins are attached and where.

Proteasome and lysosome

Proteasome or lysosomeThere are two main systems for destroying misfolded proteins: proteasomes and lysosomes. Proteasomes are described above.

In addition to proteasomes, there is another important structure that breaks down misfolded proteins: the lysosome. Lysosomes are membrane-bound compartments, filled with destructive enzymes, present in eukaryotic cells. Lysosomes are responsible for autophagy, the process by which cells break down some of their own components in a recycling process. Lysosomes also function in phagocytosis, in which a cell engulfs material from outside and brings it into the lysosome for destruction. In animal cells, phagocytosis is an essential process of the immune system.

Chaperones can target misfolded proteins to lysosomes for destruction, in a process called chaperone-mediated autophagy. More than just a part of quality control, this process helps determine helps control cell activities by balancing the competing functions of different protein and the cell's need for resources.

Overall, the protein quality control system is essential for every cell, but it doesn't always work perfectly. Quality control systems can be overwhelmed if cellular stress creates misfolded proteins and those proteins aggregate faster than they can be destroyed. Many neurodegenerative diseases are connected to misfolding & aggregation, and all such diseases involve failures of quality control systems.

Why does heat kill cells?

Heat denatures proteins

heat kills cellsIn order to understand more about the role of protein misfolding and aggregation, a group of researchers asked a simple question: why does heat kill cells?

Heat denatures proteins because the weak bonds that make up secondary and tertiary structure are easily broken by thermal energy. When cells are overheated, proteins unfold, then misfold and form aggregates, which can't be re-folded. In other words, if you cook some cells, they're not going to un-cook themselves.

 

A small amount of denaturation kills a cell

heat kill graph

The surprising finding from this research was that cells are killed by heat when only a small percentage of proteins becomes denatured. This graph shows that cell death happens when less than 10% of proteins have been denatured.

The insurmountable problem for the cell isn't the total number of denatured proteins; it's that there are particular proteins that are critical for survival, and these proteins are the first to become denatured.

This seems strange: if these proteins are so critical to survival, why are they so delicate? Why hasn't natural selection favored tougher versions of these proteins?

Interactome

interactomeThe interactome, or protein interaction map, for E. coli cells explains why. In this diagram, each dot represents a specific protein. Each line represents an interaction between two proteins. For example, a protein might modify another protein, pushing it to take an active or inactive conformation. The overall activities of the cell depend on which proteins are active at any given moment, depending on all these interactions.

Some proteins interact with multiple partners, while others, performing specialized jobs, interact with only a few. Proteins that interact with multiple partners are critical, because they regulate the cell's activities. If these proteins become denatured, the cell's regulatory mechanisms are disrupted and the cell dies. Each critical regulatory protein appears as a hub, with multiple lines radiating out from it.

The color of each dot represents the temperature corresponds to the temperature at which it becomes denatured. The blue dots are proteins that are denatured at relatively low temperatures; in other words, they're the least stable proteins.

The diagram shows that some of the most important regulatory proteins — those with the most interactions — are also the proteins that are easiest to denature.

This study points to an explanation of why this is the case. Proteins that interact with multiple other protein partners must have flexible shapes to accommodate those varying interactions. Such proteins are said to be intrinsically disordered, meaning that they have no fixed conformation. They change their conformation according to context. In turn, this makes them easy to denature. Proteins must be flexible to do their jobs, and this makes them vulnerable to denaturation

p53

p53 The protein p53 is an example of an intrinsically disordered regulatory protein. If you have an outstanding memory, you might recall that p53 was briefly mentioned on the bats & viruses page, as part of the DNA damage response pathway. In response to DNA damage, p53 turns on genes for DNA repair, stopping cell proliferation, or even inducing apoptosis (cell death). It is both a transcription factor and a tumor suppressor, two functions you'll learn more about later this quarter. The p53 protein is multifunctional & intrinsically disordered. Cells lacking functional p53 genes and proteins are very likely to become cancerous.

You'll see p53 again in the context of cell cycle checkpoints.

Summary

Cells constantly synthesize proteins, some of which will become misfolded. Survival depends on a balance of synthesis, proper folding, and removal of misfolded proteins. This balance is sometimes called proteostasis, for homeostasis of the proteome.

Review

Terms & concepts

  • Aggregation (the formation of aggregates)
  • Chaperone
  • Denaturation
  • Energy landscape of protein folding
  • Hydrophilic and hydrophobic regions of proteins
  • Interactome
  • Intrinsically disordered proteins
  • Lysosome
  • Native conformation
  • p53: how can it have multiple functions?
  • Proteasome
  • Proteostasis
  • Proteome (the complete set of proteins made by a cell or an organism)
  • Translation (we'll look at this in detail later)
  • Ubiquitin and ubiquitination (also called ubiquination or ubiquitylation)
  • Quality control: refold or destroy
  • Why heat kills cells

Vocabulary Note

Proteasome vs. proteome: You'll see both these words in biology. They look similar at first, but are completely different. To understand why, it helps to look closely at the words themselves.

  • Proteome: The complete set of proteins made by a cell. This word was created as an analogy to genome, the complete set of DNA sequences in a cell.
  • Proteasome: A structure that destroys proteins. The word is based on "protease," which refers to enzymes that break down proteins. Enzymes are proteins that catalyze chemical reactions. Most enzymes are named for what they do, and have names that end in -ase. The ending "-some" in this case means body or structure (as in lysosome, ribosome, or somatic cells). So the proteasome is a structure that catalyzes protein destruction.

You're going to encounter a lot of new words in biology; it's often helpful to understand where they come from in order to remember what they mean.

Review Questions

  1. Why do misfolded proteins form aggregates?
  2. Why are exposed hydrophobic regions of polypeptides unstable?
  3. How are cytosolic proteins different from membrane proteins? (Include hydrophobic side chains in your answer.)
  4. How can protein folding or misfolding be related to diseases?
  5. Describe the four levels of protein structure. What kinds of bonds or interactions are responsible for each?
  6. What controls the 3-dimensional shape of proteins in cells?
  7. How does protein quality control work? How could it fail?
  8. What are the two main ways to destroy denatured proteins in cells?
  9. What kinds of diseases are connected to protein misfolding?
  10. Why do proteins do most of the work in cells? Why not polysaccharides or nucleic acids?
  11. Look closely at the interactome, or map of protein interactions. What does each dot represent? What do the lines represent? What do the colors of the dots represent?
  12. Consider a protein like p53, which interacts with many different protein partners to control multiple processes within the cell. Do you think this protein would be stable at relatively high temperatures, or easily denatured even at relatively low temperatures?

References & further reading

You don't need to use any of the sources here. I'm offering you some alternative presentations that might aid your learning, and some opportunities for deeper reading if you're interested. The quiz on this topic will be based on the material shown on this page.

Videos

What is a protein?. 7 min; from PDB-101. Excellent overview of protein structure; in particular, the first 4 minutes show the levels of protein structure (which will be on the quiz!). PDB-101 is an outstanding site about proteins; I highly recommend it if you’re interested in molecules.

Protein Folding from AskaBiologist. Brief graphic look at folding.

Protein Folding Mechanism from Hussain Biology. 8 minutes.

Easy reading

Chaperones from PDB-101. Graphic overview of chaperones, with some simple examples. Includes a great explanation of how the GroEL-GroES chaperone works.

Ubiquitin (PDB-101) and The Nobel Prize in Chemistry, 2004: Popular Information. These two articles give the story on ubiquitin, for which the Nobel Prize was awarded.

The protein folding problem

For decades, this has been one of the hard problems in basic science: predicting conformation based on amino acid sequences. Recently, there have been some important advances in this area.

The protein folding revolution video from Science Magazine. Describes the process of using software to try to figure out how proteins will fold. The accompanying article, This protein designer aims to revolutionize medicines and materials (Links to an external site.) describes the process of designing synthetic proteins for biological functions.

AlphaFold: a solution to a 50-year-old grand challenge in biology from DeepMind. Describes an advanced AI-based approach to the remarkably difficult problem of predicting how a protein will fold, based on its primary structure (which in turn can be predicted from DNA sequences).

Why heat kills cells

How heat kills cells. Greenwood, 2017, Quanta. (Reprinted in The Atlantic). Detailed but readable, this article describes the heat and misfolding research I briefly mentioned above. Contains some interesting insights into aggregation. Overall, this article exemplifies the way that modern biologists tend to look at proteins: not as entities functioning in isolation, but as parts of networks. This non-technical article is based in part on the research article listed below.

Cell-wide analysis of protein thermal unfolding reveals determinants of thermostability (abstract only) Leuenberger et al., 2017, Science Magazine. The research article.

Scientific literature

The Biology of Proteostasis in Aging and Disease (Links to an external site.). Labbadia, 2015. Excellent review for anyone who wants to go deep into this topic. "We explore emerging evidence that disease susceptibility arises from early changes in the composition and activity of the proteostasis network...."

 

A- A A+