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Orders of protein structure

Orders of protein structure: primary, secondary, tertiary, and quaternary. Alpha helix and beta pleated sheet.


Have you ever wondered why egg whites go from clear to opaque when you fry an egg? If so, this section is for you!
Egg whites contain large amounts of proteins called albumins, and the albumins normally have a specific 3D shape, thanks to bonds formed between different amino acids in the protein. Heating causes these bonds to break and exposes hydrophobic (water-hating) amino acids usually kept on the inside of the protein1,2. The hydrophobic amino acids, trying to get away from the water surrounding them in the egg white, will stick to one another, forming a protein network that gives the egg white structure while turning it white and opaque. Ta-da! Thank you, protein denaturation, for another delicious breakfast.
As we mentioned in the last article on proteins and amino acids, the shape of a protein is very important to its function. To understand how a protein gets its final shape or conformation, we need to understand the four levels of protein structure: primary, secondary, tertiary, and quaternary.

Primary structure

The simplest level of protein structure, primary structure, is simply the sequence of amino acids in a polypeptide chain. For example, the hormone insulin has two polypeptide chains, A and B, shown in diagram below. (The insulin molecule shown here is cow insulin, although its structure is similar to that of human insulin.) Each chain has its own set of amino acids, assembled in a particular order. For instance, the sequence of the A chain starts with glycine at the N-terminus and ends with asparagine at the C-terminus, and is different from the sequence of the B chain.
Image of insulin. Insulin consists of an A chain and a B chain. They are connected to one another by disulfide bonds (sulfur-sulfur bonds between cysteines). The A chain also contains an internal disulfide bond. The amino acids that make up each chain of insulin are represented as connected circles, each with the three-letter abbreviation of the amino acid's name.
image credit: OpenStax Biology.
The sequence of a protein is determined by the DNA of the gene that encodes the protein (or that encodes a portion of the protein, for multi-subunit proteins). A change in the gene's DNA sequence may lead to a change in the amino acid sequence of the protein. Even changing just one amino acid in a protein’s sequence can affect the protein’s overall structure and function.
For instance, a single amino acid change is associated with sickle cell anemia, an inherited disease that affects red blood cells. In sickle cell anemia, one of the polypeptide chains that make up hemoglobin, the protein that carries oxygen in the blood, has a slight sequence change. The glutamic acid that is normally the sixth amino acid of the hemoglobin β chain (one of two types of protein chains that make up hemoglobin) is replaced by a valine. This substitution is shown for a fragment of the β chain in the diagram below.
Image of normal and sickle cell mutant hemoglobin chains, showing substitution of valine for glutamic acid in the sickle cell version.
Image modified from OpenStax Biology.
What is most remarkable to consider is that a hemoglobin molecule is made up of two α chains and two β chains, each consisting of about 150 amino acids, for a total of about 600 amino acids in the whole protein. The difference between a normal hemoglobin molecule and a sickle cell molecule is just 2 amino acids out of the approximately 600.
A person whose body makes only sickle cell hemoglobin will suffer symptoms of sickle cell anemia. These occur because the glutamic acid-to-valine amino acid change makes the hemoglobin molecules assemble into long fibers. The fibers distort disc-shaped red blood cells into crescent shapes. Examples of “sickled” cells can be seen mixed with normal, disc-like cells in the blood sample below.
Image credit: OpenStax Biology modification of work by Ed Uthman; scale-bar data from Matt Russell.
The sickled cells get stuck as they try to pass through blood vessels. The stuck cells impair blood flow and can cause serious health problems for people with sickle cell anemia, including breathlessness, dizziness, headaches, and abdominal pain.

Secondary structure

The next level of protein structure, secondary structure, refers to local folded structures that form within a polypeptide due to interactions between atoms of the backbone. (The backbone just refers to the polypeptide chain apart from the R groups – so all we mean here is that secondary structure does not involve R group atoms.) The most common types of secondary structures are the α helix and the β pleated sheet. Both structures are held in shape by hydrogen bonds, which form between the carbonyl O of one amino acid and the amino H of another.
Images showing hydrogen bonding patterns in beta pleated sheets and alpha helices.
Image credit: OpenStax Biology.
In an α helix, the carbonyl (C=O) of one amino acid is hydrogen bonded to the amino H (N-H) of an amino acid that is four down the chain. (E.g., the carbonyl of amino acid 1 would form a hydrogen bond to the N-H of amino acid 5.) This pattern of bonding pulls the polypeptide chain into a helical structure that resembles a curled ribbon, with each turn of the helix containing 3.6 amino acids. The R groups of the amino acids stick outward from the α helix, where they are free to interact3.
In a β pleated sheet, two or more segments of a polypeptide chain line up next to each other, forming a sheet-like structure held together by hydrogen bonds. The hydrogen bonds form between carbonyl and amino groups of backbone, while the R groups extend above and below the plane of the sheet3. The strands of a β pleated sheet may be parallel, pointing in the same direction (meaning that their N- and C-termini match up), or antiparallel, pointing in opposite directions (meaning that the N-terminus of one strand is positioned next to the C-terminus of the other).
Certain amino acids are more or less likely to be found in α-helices or β pleated sheets. For instance, the amino acid proline is sometimes called a “helix breaker” because its unusual R group (which bonds to the amino group to form a ring) creates a bend in the chain and is not compatible with helix formation4. Proline is typically found in bends, unstructured regions between secondary structures. Similarly, amino acids such as tryptophan, tyrosine, and phenylalanine, which have large ring structures in their R groups, are often found in β pleated sheets, perhaps because the β pleated sheet structure provides plenty of space for the side chains4.
Many proteins contain both α helices and β pleated sheets, though some contain just one type of secondary structure (or do not form either type).

Tertiary structure

The overall three-dimensional structure of a polypeptide is called its tertiary structure. The tertiary structure is primarily due to interactions between the R groups of the amino acids that make up the protein.
R group interactions that contribute to tertiary structure include hydrogen bonding, ionic bonding, dipole-dipole interactions, and London dispersion forces – basically, the whole gamut of non-covalent bonds. For example, R groups with like charges repel one another, while those with opposite charges can form an ionic bond. Similarly, polar R groups can form hydrogen bonds and other dipole-dipole interactions. Also important to tertiary structure are hydrophobic interactions, in which amino acids with nonpolar, hydrophobic R groups cluster together on the inside of the protein, leaving hydrophilic amino acids on the outside to interact with surrounding water molecules.
Finally, there’s one special type of covalent bond that can contribute to tertiary structure: the disulfide bond. Disulfide bonds, covalent linkages between the sulfur-containing side chains of cysteines, are much stronger than the other types of bonds that contribute to tertiary structure. They act like molecular "safety pins," keeping parts of the polypeptide firmly attached to one another.
Image of a hypothetical polypeptide chain, depicting different types of side chain interactions that can contribute to tertiary structure. These include hydrophobic interactions, ionic bonds, hydrogen bonds, and disulfide bridge formation.
Image modified from OpenStax Biology.

Quaternary structure

Many proteins are made up of a single polypeptide chain and have only three levels of structure (the ones we’ve just discussed). However, some proteins are made up of multiple polypeptide chains, also known as subunits. When these subunits come together, they give the protein its quaternary structure.
We’ve already encountered one example of a protein with quaternary structure: hemoglobin. As mentioned earlier, hemoglobin carries oxygen in the blood and is made up of four subunits, two each of the α and β types. Another example is DNA polymerase, an enzyme that synthesizes new strands of DNA and is composed of ten subunits5.
In general, the same types of interactions that contribute to tertiary structure (mostly weak interactions, such as hydrogen bonding and London dispersion forces) also hold the subunits together to give quaternary structure.
Flowchart depicting the four orders of protein structure.
Image modified from OpenStax Biology's modification of work by the National Human Genome Research Institute.

Denaturation and protein folding

Each protein has its own unique shape. If the temperature or pH of a protein's environment is changed, or if it is exposed to chemicals, these interactions may be disrupted, causing the protein to lose its three-dimensional structure and turn back into an unstructured string of amino acids. When a protein loses its higher-order structure, but not its primary sequence, it is said to be denatured. Denatured proteins are usually non-functional.
For some proteins, denaturation can be reversed. Since the primary structure of the polypeptide is still intact (the amino acids haven’t split up), it may be able to re-fold into its functional form if it's returned to its normal environment. Other times, however, denaturation is permanent. One example of irreversible protein denaturation is when an egg is fried. The albumin protein in the liquid egg white becomes opaque and solid as it is denatured by the heat of the stove, and will not return to its original, raw-egg state even when cooled down.
Researchers have found that some proteins can re-fold after denaturation even when they are alone in a test tube. Since these proteins can go from unstructured to folded all by themselves, their amino acid sequences must contain all the information needed for folding. However, not all proteins are able to pull off this trick, and how proteins normally fold in a cell appears to be more complicated. Many proteins don’t fold by themselves, but instead get assistance from chaperone proteins (chaperonins).

Explore outside of Khan Academy

Do you want to learn more about protein structure and folding? Check out this scrollable interactive from LabXchange.
Do you want to learn more about the effect of temperature on protein folding? Check out this interactive image from LabXchange.
LabXchange is a free online science education platform created at Harvard’s Faculty of Arts and Sciences and supported by the Amgen Foundation.

Want to join the conversation?

  • aqualine ultimate style avatar for user Kam
    When we digest meals rich in protein, are we denaturing the proteins and "re-naturing" them through the help of chaperonins, or breaking it down to it's base amino acids and re-creating proteins using those?
    (65 votes)
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    • piceratops ultimate style avatar for user Darmon
      Oftentimes, we are breaking them down to their amino acid bases and creating new proteins. This is because many of the proteins that are found in the human body are not obtained directly from food, rather we need certain proteins in the food so we can use their amino acids to build the necessary proteins. The human body does not produce all 23 required amino acids, so we need to get them from our food, by eating proteins that contain those amino acids. :)
      (95 votes)
  • male robot hal style avatar for user sewerrat616
    What is the N-terminus and C-terminus mentioned in primary structure?
    (19 votes)
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  • aqualine ultimate style avatar for user Sean Collin
    In the Primary Structure section when they're talking about Hemoglobin and sickle cell:

    "What is most remarkable to consider is that a hemoglobin molecule is made up of two α chains and two β chains, each consisting of about 150 amino acids, for a total of about 600 amino acids in the whole protein."

    What do they mean by two Alpha chains and two Beta chains? Do they mean two polypeptides in an Alpha helix shape and two polypeptides in a Beta pleated sheet shape? Or are they just calling two of the polypeptides "alpha" because they're both identical and the other two "beta" because they're also identical.

    I thought polypeptides could contain both Alpha helix's and Beta pleated sheets, if so you won't be able to call one polypeptide chain specifically one or the other.
    (15 votes)
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    • female robot grace style avatar for user tyersome
      Your second guess is correct – the two alpha chains are identical (as are the two beta chains).

      This nomenclature is confusing and the designation of alpha and beta chains has nothing to do with alpha helices and beta sheets!

      In fact, alpha- and beta-hemoglobins have very similar structures both of which are dominated by alpha-helices and have no beta sheet at all (see for example: http://www.rcsb.org/pdb/explore/jmol.do?structureId=2HHB&bionumber=1).

      And yes, many proteins have both alpha helices and beta sheets.
      (2 votes)
  • blobby green style avatar for user juvaldez50
    It goes in depth about the structures, but lacks in explaining function. Besides holding other primary structure proteins, what does a tertiary and quaternary structure even do?
    (8 votes)
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    • duskpin tree style avatar for user Katherine Lu
      The function of tertiary and quaternary structure varies depending on type of protein, but in enzymes, the specific shape and configuration of the protein allows the formation of active sites. For example, catalase, an enzyme that breaks hydrogen peroxide into hydrogen and oxygen gas, has its proteins and amino acids configured in a certain way to create an area where the charges are so strong that spontaneous reaction occurs, by lowering the energy needed to break intermolecular bonds.
      (12 votes)
  • leaf green style avatar for user Alyssia
    A question that came to mind as I was reading this and watching the previous videos is:
    How do we know what amino acids look like? For example, we learned that there are two amino acids switched out which causes sickle cell anemia. How did we learn that and how do we know which amino acids that is?
    We don't see the letters "C", "H", "S", "N", etc when we look through microscopes at proteins, so how did we get so advanced to understand protein interactions? It's fascinating!! Thanks!
    (9 votes)
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    • female robot grace style avatar for user tyersome
      This is a great question, but actually quite complicated so I'm not going to try to give a complete answer — I have given some useful links below if you wish to learn more.

      Each amino acid has unique chemical properties that can be used to tell them apart.
      There are also methods that have been developed to remove amino acids one at a time.
      By combining theses techniques it is possible to directly determine protein sequences.

      There are many different techniques for directly determining protein sequences — this wikipedia article is a decent introduction:

      The very first protein sequence (bovine insulin) was determined by Fredrick Sanger in 1951-2 (note that this was more than a decade before the first nucleotide sequence).

      However, it is now relatively rare to directly determine protein sequence!
      Instead, since it has been worked out (mostly) how DNA codes for protein, we usually infer the protein sequence from the DNA sequence. This is because it is now much easier to sequence DNA.

      Note that because of processes such as the post-translational modifications to proteins we still need protein sequencing and I believe that we currently rely too heavily on DNA sequencing.
      (9 votes)
  • duskpin ultimate style avatar for user Kaitlyn Melody
    I am slightly confused with secondary structures. If the backbone determines the structure, how is it different from the tertiary structures? In other words, what are the main difference between each structure?
    (5 votes)
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    • orange juice squid orange style avatar for user briancsherman
      The secondary structure is formed by hydrogen bonds between carbonyl and amino groups that make up the polypeptide backbone and causes the molecule to either bend and fold (beta pleated sheet) or spiral around (helicase).

      The tertiary structure is formed by many different bonds between R groups that make up the side chains, that make the strand of molecule bend and loop around in a more complicated three dimensional form.
      (9 votes)
  • aqualine ultimate style avatar for user Sean Collin
    In the last paragraph they begin to talk about Proteins "folding" but they don't explain what this means.
    (4 votes)
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  • aqualine ultimate style avatar for user Jack Lance
    In the subsection on Tertiary Structure, it is mentioned that Disulfide bonds are much stronger than the other types of bonds contributing to Tertiary Structure of proteins. What is it about these S-S linkages that makes them so strong? And in relation to that: What determines whether or not an ionic/covalent bond is strong or weak?
    (5 votes)
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  • aqualine ultimate style avatar for user Amirhossein Alesheikh
    In the sickle cell anemia example, is it the case that a key amino acid difference may cause catastrophic results or is it the case for that all amino acids are that important and changing any of them may cause similar catastrophic results?
    (4 votes)
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    • piceratops seed style avatar for user RogerP
      Sickle cell anaemia is caused by a single point mutation of the gene coding for haemoglobin. As a result, a hydrophobic amino acid (valine) replaces the usual hydrophilic amino acid (glutamic acid). This very small change in sequence affects the outside of the protein and results in haemoglobin molecules sticking together, changing the shape of the cell.

      There are lots of other known mutations in the haemoglobin gene, some which affect health and others which have no impact.

      In summary, for any particular protein, some amino acids are far more important than others.
      (4 votes)
  • leafers sapling style avatar for user connerjamesstarnes
    What duos it mean by denatured?
    (4 votes)
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    • winston baby style avatar for user Mason Smith
      In biochemistry, denaturation is a process in which proteins or nucleic acids lose the quaternary structure, tertiary structure, and secondary structure which is present in their native state, by application of some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent, agitation and radiation or heat. If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death.

      But in the "Denaturation and protein folding", I think they were talking about if the pH or temperature of a proteins environment was to change, the shape of the protein (I think) would change also, and when that happens, the protein is going to turn back into an unstructured string of amino acids. And when a protein loses it's high order structure (but not the primary one) it is called denatured.
      (3 votes)