Quaternary Level of Protein Structural Organization

Quaternary structure or Quaternary level of protein structural organization, is the structure that results of  the assembly of several polypeptide to make an unique functional protein, stabilized through several noncovalent interactions between the R side chain of amino acids from different peptide chains.


The non covalent interactions that maintain this structure are the same non covalent interactions that maintain the tertiary structure: Hydrogen bonds, Ionic interactions, Hydrophobic attractions and Van der Waals Forces.


Based on the definition we gave above, not all proteins show a quaternary level of organization. For having a quaternary structure:

a)     The protein should be formed by more than one peptide chain.

b)     These chains can not be attached by covalent bonds among them.


Some examples for clarifying the concept:


Myoglobin is formed by a single peptide chain and a hem group. Since Myoglobin is formed by just one peptide chain, it does not show quaternary structure.


Insulin, for example, is formed by two peptide chains, but since these two chains are linked by disulfide linkage, insulin does not qualify as a protein with quaternary structure.


Hemoglobin is formed by four peptide chains (and four Hem groups) that are forming a unique functional protein. These peptide chains are associated through non covalent bonds between their lateral chains: Hemoglobin is the typical example of a protein with quaternary structure.






The quaternary structure of a protein is intimately related to the feature of Allostery or Allosterism


It’s the property of some proteins to change its conformation and activity (it changes from a native conformation to  a different native conformation), when interact specifically with some ligands.


In the case of enzymes, the ligand binds at a site different to the active site, that is called allosteric site. This change in conformation results in a change in the activity of the enzyme.


Characteristics of the Allosteric proteins


         All reach the quaternary level

         They have two different but native structures

         Each structure has a different functionality

         The two native structures are in equilibrium

         The equilibrium between the two structures is displaced when the protein interact with specific ligands

         The interaction with the ligand takes place in specific sites of the protein known as allosteric sites


Hemoglobin, mentioned above,  is a typical example of a protein with quaternary structure that shows allosterism.  


Hemoglobin has two different native structures:

– Oxyhemoglobin (Relaxed structure).

– Deoxyhemoglobin (Tense or Taught structure).


These different native structures have different functionality;

– The Relaxed (R ) structure has high O2 affinity.

– The taught or tense (T) structure has low O2 affinity.


These two forms are in equilibrium:


R ß——à T


Presence or absence of oxygen changes the conformation: when Hemoglobin binds to Oxygen, the equilibrium between these two kinds of structures is displaced to the R form; when Oxygen is released, the equilibrium is displaced to the T form.


These two animations reflect the change of Hemoglobin structure as a result of Oxygen binding:







Allosterism is also an important physiological mechanism of regulation of enzyme activity.



For more information about these topics, visit the following sites:


Berg, J.M.; Tymoczko, J.L. Stryer, L.: Biochemistry, 5t. Edition:


Quaternary Structure: Polypeptide chains can assemble into multisubunits structure.


Oxygen binding markedly changes the quaternary Structure of Hemoglobin








The Medical Biochemistry page


Quaternary structure





Allosteric enzymes




Supersecondary Structures (Motifs) and Domains.


We can define Supersecondary structures as combinations of alpha-helices and beta-structures connected through loops, that form patterns that are present in many different protein structures. These folding patterns are stabilized through the same kind of linkages than the tertiary level. Sometimes the term “motif” is used to describe these supersecondary structures.


These structures can be relatively simples, as alpha-alpha (two alpha helixes linked by a loop), Beta-Beta (two beta-strands linked by a loop), Beta-alpha-Beta (Beta-strand linked to an alpha helix that is also linked to other beta strand, by loops) or more complexes structures, like the Greek key motiv or the beta-barrel.

Greek Key motif

Greek Key motif



Beta-barrel motif

Beta-barrel motif


It is very interesting in these motifs that these repetitive structures can be very different in their primary structure and they can be present in very different proteins.  Some proteins have no supersecondary structures.




They are stable, independently folding, compact structural units  within a protein, formed by segments of the polypeptide chain, with relative independent structure and function distinguishable from other regions and stabilized through the same kind of linkages than the tertiary level.


Following this definition, in this representation of Pyruvate kinase, it is possible to distinguish three domains:


Protein domains may be considered as elementary units of protein structure and evolution, capable, to some extent, of folding and functioning autonomously. A domain sometimes contain motifs, sometimes don’t.


The tertiary structure of many proteins is built from several domains


Domain Functions:


Often each domain has a separate function to perform for the protein, such as:


– Bind a small ligand


– Spanning the plasma membrane (transmembrane proteins)


– Contain the catalytic site (enzymes)


– DNA-binding (in transcription factors)


– Providing a surface to bind specifically to another protein


In some (but not all) cases, each domain in a protein is encoded by a separate exon in the gene encoding that protein.



More information:


Supersecondary structures






Protein domains



Tertiary Level of Protein Structural Organization


A very clear and concise definition of tertiary structure appears in Mark’s Medical Biochemistry, which defines this structural level as “the folding pattern of the secondary structure into a three-dimensional conformation”.


Another way of defining it is based on the linkages that maintain this structural level. The tertiary structure of proteins is usually defined as the spatial conformation of the protein stabilized through several interactions between the R side chains of distant amino acids residues. Distant means that they can be very apart in the sequence, but because of the molecule folding, their lateral chains can interact through their functional groups. These interactions stabilize the spatial conformation of the protein.


Hydrophobic interactions are very often the driven force that allows that lateral chain of distant amino acids becomes next each other. In an aqueous environment, non polar lateral chains of amino acids in a peptide or protein tend to cluster together, as a result of the hydrophobic forces. It allows other kinds of interaction between the “new neighbors” amino acids.


The main interactions that maintain the spatial conformation of the proteins are:


Hydrophobic interactions (already mentioned)

Hydrogen bonds between the R side chain of distant amino acids

Ionic interactions

Disulfide bridges


Hydrophobic interactions


Already described. The amino acids with non polar lateral chain that can be attracted among them by hydrophobic forces are Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tyrosine and Tryptophan


Hydrogen bonds between the side chains (R) of distant amino acids


Hydrogen bonds, as we know, are established between very electronegative atom and Hydrogen bonded to Fluoride, Oxygen or Nitrogen.


Hydrogen bonds

Hydrogen bonds


These bonds can be established between molecules (like in the former example) or between parts of the same molecule, like occurs in proteins.


The amino acids whose lateral chains can form Hydrogen bonds among them that stabilize the tertiary structure are those that have Oxygen or Nitrogen in their lateral chain: 


Serine (hydroxyl group)

Threonine (hydroxyl group)

Tyrosine (hydroxyl group)

Glutamate (carboxyl group)

Glutamine (carbamide group)

Aspartate (carboxyl group)

Asparagine (carbamide group)

Lysine (amine group)

Arginine (amidine group)

Histidine (imidazol group)


(The hydrogen bonds between the lateral chains of amino acids, that stabilize the tertiary structure, should not be confounded with the Hydrogen bonds between elements of the peptide bonds, which are characteristics of the secondary structure)


Ionic interactions


Charged lateral chains of amino acids can interact with charged lateral chains of other amino acids. Amino acids with charged lateral chain are Glutamate, Aspartate, Lysine, Arginine and Histidine. If the amino acids whose lateral chains are interacting  have the same charges, the interaction results in repulsion, so the parts of the peptide chain where the amino acids are located, will separate; if they have different charges, the portions of the peptide chain were they are located will be attracted.



Interaction between aspartate and glutamate (both with negative lateral chain).

Interaction between two of the following: Lysine, Arginine, Histidine (all of them with positively charged lateral chain)



Interaction between glutamate or aspartate (negatively charged lateral chains) with Lysine, Arginine or Histidine (positively charged lateral chains).


Disulfide bridges


Disulfide bond

Disulfide bond



As you know, cystein has an R-SH group in it lateral chain





Disulfide bridges are formed when two residues of cysteine interact between them, experimenting an oxidation of the sulfhydril groups. As a result both cysteines become linked, forming a cystine residue.





Disulfide bridges are very strong bonds, that can be formed between different parts of the same peptide chain (intrachain bonds) if the cystein residues that form the disulfide bridge are in the same chain, or they can form interchain bonds, maintaining together different peptide chains, if the cysteine residues that form this linkage are present in different peptide chains.


Disulfide bonds are very frequent in proteins. Insulin has intermolecular and intramolecular disulfide bridges. Immunoglobulin chains are maintained together by disulfide bridges. The following diagram represent an immunoglobulin, whose Heavy and Light chains are maintained together by disulfide bridges (represented in red)



Last but not least: the tertiary structure of a protein is intimately related to its function. Proteins that lost the native tridimensional conformation lost their functions. The main causes of losing a native conformation are denaturalization or a mutation (recall that the primary structure –amino acid sequence -determines the higher structures of the protein).


More information about Tertiary structure of proteins can be found at:










Secondary Structure of Proteins


As described before, we can distinguish in proteins four different organizational levels:



The secondary structure or secondary level of organization has been defined as the conformation present in a local region of the polypeptide or protein, stabilized through hydrogen bonds between the elements of the peptide bond.


The organized secondary structures are maintained by Hydrogen bonding between different peptide groups, it means, between the N-H group of one peptide bond and a C=O group of another peptide bond.




Pauling and Corey, when studying the possible secondary structures of proteins, described that the main structures forming part of this level of organization should have the following characteristics (Pauling and Corey postulates):


         All amino acids belong to the steric L series

         The peptide group is planar and in trans configuration

         Every carbonyl oxygen and amide nitrogen are involved in hydrogen bond formation

         Rotation of the molecule only around the alpha-carbon atoms

         The hydrogen-bonded hydrogens lie close to a line joining the oxygen and nitrogen atoms involved in formation of the bond

         The R side chain of amino acids are not involved in the structure


The structures that are considered in this secondary level of organization include:




Beta-pleated sheet




Beta -turns (aka Beta-bends aka hairpin bends)


Nonrepetitive secondary structure


Alpha helix


It’s the secondary level of protein organization in which the polypeptide backbone is tightly wound around an imaginary axis as a spiral structure. (Helicoidal arrangement of the peptide chain)


In this clip, Linus Pauling describes how he discovered the alpha-helix:


Structural features of the Alpha-Helix:


– There are 3.6 amino acids per turn of the helix.

– Each peptide bond is trans and planar

– N-H groups of all peptide bonds point in the same   direction, which is roughly parallel to the axis of the    helix

– C=O groups of all peptide bonds point in the opposite direction, and also parallel to the axis of the helix

– The C=O group of each peptide bond is hydrogen bonded to the N-H group of the peptide bond four amino acid units away from it

– All R- groups point outward from the helix


Alpha-helix stability is affected by different factors, which include:


1. – electrostatic interaction between successive amino acids with R charged groups.

2. – the bulkiness of adjacent R groups

3. – Interactions between R groups spaced 3 or 4 residues apart.

4. – Occurrence of Pro residues.


About 1/4th of all amino acid residues in polypeptides are found in alpha-helices, the exact fraction varying greatly from one protein to the next

Observe in this example the alpha helix structures in calmoludin:


Beta pleated sheet



This secondary structure has been defined as the secondary level of protein organization in which the backbone of the peptide chain (Beta-strands)  is extended into a zigzag arrangement resembling a series of pleats, with the peptide bonds organized in planes of alternating slopes (alternating ascending and descending direction). The Beta pleated sheet can be formed between two peptide chains or between different segments of the same peptide chain.



Characteristics of the Beta-pleated sheet include;


1. – Each peptide bond is planar and has the trans conformation

2.- The C=O and N-H groups of peptide bonds from adjacent chains point toward each other and are in the same plane so that hydrogen bonding is possible between them

3.- All R- groups on any one chain alternate, first above, then below the plane of the sheet, etc.


There are two kinds of Beta pleated sheets:


Antiparallel: when the adjacent polypeptide chains run in opposite direction



Parallel: Adjacent polypeptide chains running in the same direction


Usually the segment of polypeptides that shows a Beta conformation are called individually beta-strands, and they are represented by an arrow pointing in the direction in which the strand runs (from the amino to the carboxyl group)


Both models are found in proteins, but the antiparallel structure is more stable than the parallel beta-sheet.

Betas conformation content in proteins is very variable: myoglobin, for example, does not show this kind of secondary structure, while 45 percent of the amino acids in chymotrypsin are part of a beta conformation.


It is important to note that not all the polypeptide chain is part of an alpha-helix or a beta conformation. There are also bends, segments irregularly coiled or forming extended stretches: Carboxypeptidase, for example, shows 38 % of the amino acids forming alpha-helix and 27 % forming beta structures, consequently around 35 % of the residues are not included in these secondary structures.


Beta turn


It’s the secondary level of protein organization which permits the change of direction of the peptide chain to get a folded structure.



They are known as well as reverse turns, hairpin bends or Omega-loops; they occur often in 5 amino acid residues or less and they lie on the protein surface because they are hydrophilic. Beta-turn loops allow for protein compaction, since the hydrophobic amino acids tend to be in the interior of the protein, while the hydrophilic residues interact with the aqueous environment.


Observe in the following figure how alpha-helix structures and Beta- conformations (beta strands represented by arrows) are linked through bents and beta turns that make this protein compact



Different amino acids favor different kind of secondary structures: while alanine, glutamate, and leucine have a propensity for being in α helices, valine and isoleucine apparently favor β strands, and glycine, asparagine, and proline tend to be present in beta-turns.


If you are interested in obtaining more information about secondary structure, this would be a very interesting article for beginning:


Eisemberg, D.

The discovery of the α-helix and β-sheet, the principal structural features of proteins





H. Jakubowski: Biochemistry On line. Protein Structure



Very good animations:






Q: About the structure of a membrane associated peptide


(P-15) A 42-amino acids peptide related to the extracellular Alzheimer amyloid deposits has the last few residues immersed in the membrane bilayer. Based on your knowledge about membrane proteins, which of the following sequences most probably identifies the last five amino acids in this 42 residue peptide?


a)     Ala-Glu-Phe-Arg


b)     Val-Val-Ile-Ala


c)      Asp-Ser-Gly-Tyr


d)     Lys-Val-His-His-Gln


e)     Asp-Val-Gly-Ser



Primary Structure of Proteins

As described in a previous post, four levels of organization have been distinguished in the structure of proteins:



The primary structure of proteins is the sequence of amino acids in a polypeptide chain, linked through peptide bonds, that form the covalent backbone of the proteins. The sequence of amino acids is read from the N-terminal amino acid to the C-terminal amino acid.


It is important to realize that two proteins that have the same amino acid composition, can have very different primary structure. These two peptides, for example (a peptide is a short amino acid chain formed by a few amino acids), are formed by the same amino acids, but their primary structure is different, since the sequence is different.





The primary structure determines the three-dimensional structure of the protein, which in turns determines its biological function. Alteration in normal primary structure of proteins can produce catastrophic results.


The peptide bond:


The peptide bond is a type of carbamide linkage.


An amide linkage is a bond between an acid and an amine. There are phosphamide linkages, if the acid is phosphoric acid, sulfamide linkages, if the acid is sulphuric, etc. A carbamide linkage in an amide linkage in which the acid that participates is a carboxylic acid.



A peptide bond is a kind of carbamide linkage, in which the carboxyl group belongs to one amino acid and the amine group belongs to other amino acid:



Features of the peptide bond


1.- It has the characteristic of a partial double bond between the carbon of the carbonyl group and the N from the amino group, so the bond order is 1.5; it means that it is not a rotating bond as single bonds, but it is rigid. It is said that the bond order is 1.5, to contrast to the bond order of 1, for single bonds, and bond order of 2 for double bonds.


Since the six atoms that form the peptide bond are in the same plane, and this bond is rigid, it would appear that it is possible to have trans and cis configuration, but in fact the configuration in the peptide bond is always trans.


Alterations in primary structure of the proteins may be unnoticed or may produce catastrophic results.


In some cases, a mutation change an amino acid for a very similar one, withour affecting higher structures or functions of the protein. It is called a Conservative Mutation. An example of this kind of mutations could be the change of an apolar amino acid for another apolar amino acid, or an acidic amino acid for another acidic amino acid, lile Asp for Glu.


In other cases, a mutation results in the change of an amino acid for other, usually a very different one, affecting higher structures or functions of the protein, ex. an apolar amino acid for a polar amino acid, or a negative charged by a positively charged, etc. This kind of mutation is called a Non-conservative mutation.


There has been described hundreds of mutations that affect hemoglobin. In some cases, the change of amino acid in the primary structure has been discovered by chance, since the mutation does not have cinical significance.


In other cases, the mutations produce important clinical signs. The most important example is Sickle cell anemia.



The sickle cell syndromes are caused by a mutation in the Beta-globin gene of hemoglobin that changes the sixth amino acid in the Beta-chain, from glutamic acid to valine


Type of Hemoglobin

Codons 5-6-7

Amino acids 5-6-7

 Hemoglobin A (normal)



Hemoglobin S (Sicklemia)





This small change in the primary structure puts a hydrophobic amino acid on the outside of the molecule. This hydrophobic area causes the attachment of adjacent hemoglobins when the globin molecule shifts in conformation following the release of oxygen.

The attached hemoglobin will form then polymeric chains that produce polymerized rods of liquid crystalline hemoglobin. These polymers stiffen the red blood cell membrane, increasing viscosity and causing dehydration. As a consequence of these changes, the characteristic sickle shape appears



Sickled cells lose the pliability needed to pass through small capillaries, and it provokes episodes of microvascular vasoocclusion and premature erythrocyte destruction (hemolytic anemia).



In fact, the discovery of the relationship between this small change in the primary structure of Hemoglobin and the signs and symptoms of Sickle Cell anemia, was a milestone in the development of Medical Biochemistry, Genetics and Molecular Biology, and introduced forever the expression “molecular disease”


More information can be found in the following links:


Primary structure of Proteins








Peptide bond



Different kinds of Hemoglobin:



Structure-function relations of human hemoglobin