Professional I Exam 2 May 2011 (Part 4)
Biochemistry: Structural organisation of proteins
Describe the structural organisation of proteins. (10 minutes)
Guide to answering the above question:
- Paraphrase the question: How is protein structure organised? What are the primary, secondary, tertiary and quaternary structure of proteins? Describe the primary, secondary, tertiary and quaternary structure of proteins.
- Outline: It is convenient to discuss protein structure in terms of four levels of increasing complexity (from primary to quaternary).
- Organise your answer: Answer primary structure first, then secondary structure, followed by tertiary structure and finally quaternary structure. Then give one or two examples of proteins and mention what structural levels they possess. The most common protein used for to answer this question is hemoglobin. You can use other proteins if you have studied their detailed structures,
- Website: protein-structures-primary-secondary-tertiary-quaternary/
PROTEINS
PRIMARY STRUCTURE
Primary structure is the sequence of amino acid residues making up the protein. Thus primary structure involves only the covalent bonds linking the amino acid residues together.
The minimum size of a protein is defined as about 50 residues; smaller chains are referred to simply as peptides. So the primary structure of a small protein would consist of a sequence of 50 or so residues. Even such small proteins contain hundreds of atoms and have molecular weights of over 5000 Daltons (Da). There is no theoretical maximum size, but the largest protein so far discovered has about 30,000 residues. Since the average molecular weight of a residue is about 110 Da, that single chain has a molecular weight of over 3 million Daltons.
SECONDARY STRUCTURE
This level of structure describes the local folding pattern of the polypeptide backbone and is stabilized by hydrogen bonds between N-H and C=O groups. Various types of secondary structure have been discovered, but by far the most common are the orderly repeating forms known as the alpha-helix and the beta-pleated sheet.
Alpha-helix
An
alpha-helix is a helical arrangement of a single polypeptide chain, like a coiled spring. In this conformation, the carbonyl and N-H groups are oriented parallel to the axis. Each carbonyl is linked by a hydrogen bond to the N-H of a residue located 4 residues further on in the sequence within the same chain. All C=O and N-H groups are involved in hydrogen bonds, making a fairly rigid cylinder. The
alpha-helix has precise dimensions: 3.6 residues per turn, 0.54 nm per turn. The side chains project outward and contact any solvent, producing a structure something like a bottle brush or a round hair brush. An example of a protein with many
alpha-helical structures is the
keratin that makes up human hair.
α-helix: springy/ flexible
Beta-sheet
The structure of a
beta-sheet is very different from the structure of an
alpha-helix. In a
beta-sheet, the polypeptide chain folds back on itself so that polypeptide strands lie side by side, and are held together by hydrogen bonds, forming a very rigid structure. Again, the polypeptide N-H and C=O groups form hydrogen bonds to stabilize the structure, but unlike the
alpha-helix, these bonds are formed between neighbouring polypeptide (b) strands. Generally the primary structure folds back on itself in either a parallel or antiparallel arrangement, producing a parallel or antiparallel
beta-sheet. In this arrangement, side chains project alternately upward and downward from the sheet. The major constituent of
silk (silk fibroin) consists mainly of layers of
beta-sheet stacked on top of each another.
β-sheet:
- Hydrogen bonding
- (e.g silk)
- High tensile strength due to hydrogen bonding
Other
There are other types of secondary structure. While the
alpha-helix and
beta-sheet are by far the most common types of structure, many others are possible. These include various
loops,
helices and
irregular conformations. A single polypeptide chain may have different regions that take on different secondary structures. In fact, many proteins have a mixture of
alpha-helices,
beta-sheets, and other types of folding patterns to form various overall shapes.
Interactions
What determines whether a particular part of a sequence will fold into one or the other of these structures? A major determinant is the interactions between side chains of the residues in the polypeptide. Several factors come into play: steric hindrance between nearby large side chains, charge repulsion between nearby similarly-charged side chains, and the presence of proline. Proline contains a ring that constrains bond angles so that it will not fit exactly into an a helix or b sheet. Further, there is no H on one peptide bond when proline is present, so a hydrogen bond cannot form. Another major factor is the presence of other chemical groups that interact with each other. This contributes to the next level of protein structure, the tertiary structure.
TERTIARY STRUCTURE
This level of structure describes how regions of
secondary structure fold together – that is, the 3D arrangement of a polypeptide chain, including
alpha-helices,
beta-sheets, and any other loops and folds. Tertiary structure results from interactions between side chains, or between side chains and the polypeptide backbone, which are often distant in sequence. Every protein has a particular pattern of folding and these can be quite complex.
Stabilisation of protein structure
Whereas secondary structure is stabilized by H-bonding, all four “weak” forces contribute to tertiary structure. Usually, the most important force is hydrophobic interaction (or hydrophobic bonds). Polypeptide chains generally contain both hydrophobic and hydrophilic residues. Much like detergent micelles, proteins are most stable when their hydrophobic parts are buried, while hydrophilic parts are on the surface, exposed to water. Thus, more hydrophobic residues such as trp are often surrounded by other parts of the protein, excluding water, while charged residues such as asp are more often on the surface.
Other forces that contribute to tertiary structure are ionic bonds between side chains, hydrogen bonds, and van der Waals forces. These bonds are far weaker than covalent bonds, and it takes multiple interactions to stabilize a structure.
There is one covalent bond that is also involved in tertiary structure, and that is the disulfide bond that can form between cysteine residues. This bond is important only in non-cytoplasmic proteins since there are enzyme systems present in the cytoplasm to remove disulfide bonds.
Visualization of protein structures
Because the 3D structures of proteins involve thousands of atoms in complex arrangements, various ways of depicting them so they are understood visually have been developed, each emphasizing a different property of the protein. Software tools have been written to depict proteins in many different ways, and have become essential to understanding protein structure and function.
Structural Domains of Proteins
Protein structure can also be described by a level of organization that is distinct from the ones we have just discussed. This organizational unit is the protein “domain,” and the concept of domains is extremely important for understanding tertiary structure. A domain is a distinct region (sequence of amino acids) of a protein, while a structural domain is an independently-folded part of a protein that folds into a stable structure. A protein may have many domains, or consist only of a single domain. Larger proteins generally consist of connected structural domains. Domains are often separated by a loosely folded region and may create clefts between them.
Tertiary Structure
- further folding and super coiling of the polypeptide
- controlled by interactions (covalent, ionic, van der Waals) between the R-groups or side chains of the amino acids
Interacts with watery environment of the cytoplasm to drive folding process.
- Gives protein a specific 3-dimensional shape and a specific function. What would happen to the protein if these bonds were broken? Denaturation
QUATERNARY STRUCTURE
Some proteins are composed of more than one polypeptide chain. In such proteins, quaternary structure refers to the number and arrangement of the individual polypeptide chains. Each polypeptide is referred to as a subunit of the protein. The same forces and bonds that create tertiary structure also hold subunits together in a stable complex to form the complete protein.
Quaternary Structure
- Two or more polypeptides interacting to form a functional protein
- Metal ions may be part of the protein structure as in
Individual chains may be identical, somewhat similar, or totally different. As examples,
CAP protein is a dimer with two identical subunits, whereas
hemoglobin is a tetramer containing two pairs of non-identical (but similar) subunits. It has 2
alpha subunits and 2
beta subunits.
 |
| Structure of hemoglobin |
Hemoglobin with Fe
2+
- Allows for very specific activity of the protein due to detailed globular shape and
- Indivisible, tensile (strong), flexible
- Responsible for skin elasticity
- 4 heme groups allows it to bind 4 oxygen molecules
- Successive oxygen binding is accelerated
- 1st oxygen is hardest to bind
Allosteric Co-operativity
- Property of quaternary structure
- Activates activity of a protein through its initial bonding
Secreted proteins often have subunits that are held together by disulfide bonds. Examples include tetrameric
antibody molecules that commonly have two larger subunits and two smaller subunits (“heavy chains” and “light chains”) connected by disulfide bonds and noncovalent forces.
In some proteins, intertwined
alpha-helices hold subunits together; these are called
coiled-coils. This structure is stabilized by a hydrophobic surface on each
alpha-helix that is created by a heptameric repeat pattern of hydrophilic/hydrophobic residues. The sequence of the protein can be represented as “
abcdefgabcdefg
abcdefg…” with positions “a” and “d” filled with hydrophobic residues such as A, V, L etc. Each a helix has a hydrophobic surface that therefore matches the other. When the two helices coil around each other, those surfaces come together, burying the hydrophobic side chains and forming a stable structure. An example of such a protein is
myosin, the motor protein found in muscle that allows contraction.
Protein Folding
How and why do proteins naturally form secondary, tertiary and quaternary structures? This question is a very active area of research and is certainly not completely understood. A folded, biologically-active protein is considered to be in its “native” state, which is generally thought to be the conformation with least free energy.
Proteins can be unfolded or “denatured” by treatment with solvents that disrupt weak bonds. Thus organic solvents that disrupt hydrophobic interactions, high concentrations of urea or guanidine that interfere with H-bonding, extreme pH or even high temperatures, will all cause proteins to unfold. Denatured proteins have a random, flexible conformation and usually lack biological activity. Because of exposed hydrophobic groups, they often aggregate and precipitate. This is what happens when you fry an
egg.
If the denaturing condition is removed, some proteins will re-fold and regain activity. This process is called “
renaturation.” Therefore, all the information necessary for folding is present in the primary structure (sequence) of the protein. During renaturation, the polypeptide chain is thought to fold up into a loose globule by hydrophobic effects, after which small regions of secondary structure form into especially favorable sequences. These sequences then interact with each other to stabilize intermediate structures before the final conformation is attained.
Many proteins have great difficulty renaturing, and proteins that assist other proteins to fold are called “
molecular chaperones.” They are thought to act by reversibly masking exposed hydrophobic regions to prevent aggregation during the multi-step folding process. Proteins that must cross membranes (eg. mitochondrial proteins) must stay unfolded until they reach their destination, and molecular chaperones may protect and assist during this process.
Protein families/Types of proteins
Proteins are classified in a number of ways, according to structure, function, location and/or properties. For example, many proteins combine tightly with other substances such as carbohydrates (“glycoproteins”), lipids (“
lipoproteins”), or metal ions (“metalloproteins”). The diversity of proteins that form from the 20 amino acids is greatly increased by associations such as these. Proteins that are tightly bound to membranes are called “membrane proteins”. Proteins with similar activities are given functional classifications. For example, proteins that break down other proteins are called proteases.
Because almost all proteins arise by an evolutionary process, ie. new ones are derived from old ones, they can be classified into families by their relatedness. Proteins that derive from the same ancestor are called “homologous proteins”. Studying the sequences of homologous proteins can give clues to the structure and function of the protein. Residues that are critical for function do not change on an evolutionary timescale; they are referred to as “conserved residues”. Identifying such residues by comparing amino acid sequences often helps clarify what a protein is doing or how it is folded. For example the proteases
trypsin and
chymotrypsin are members of the “serine protease” family; so-named because of a conserved serine residue that is essential to catalyze the reaction. Trypsin and chymotrypsin contain very similar folding patterns and reaction mechanisms. Recognizing a pattern of conserved residues in protein sequences often allows scientists to deduce the function of a protein.
Relevant diagram:
PowerPoints
