Protein structure

Question 1

Summarize the structure of amino acids and their basic properties

Answer 1

Amino acids join together to form proteins

• Zwitterions
• R-group determines effect of amino acid

DEF Protein:

• organic compounds
• composed of one or more chains of amino acids and
• forming an essential part of all living organisms

Amino acids are made up off central carbon atom that is bound to:

• hydrogen
• amino group (NH2, or NH3+)
• carboxyl group (COOH or COO-)
• ’R group”/side chain – this varies per amino acid and determines the function of it (and the name)
• 20 variations are essential for life, humans can synthesise 11 of these and gets the rest premade from digested proteins

Amino acids in solution at neutral pH exist predominantly as dipolar ions (also called zwitterions). In the dipolar form, the amino group is protonated (—NH3+) and the carboxyl group is deprotonated (—COO−).

They are zwitterions – a molecule were one part of positive and another is negative, due to addition/removal of proton

• the addition of removal is determined by how many protons there are (pH)
• the isoelectric point is the pH where overall there is no charge

Chirality of amino acids

• The central Ca carbon atom is a chiral centre i.e. it has four different substituents bound to it
• The same amino acid can have 2 different 3D structures
• This gives rise to optical isomers (enantiomers) of each amino acids each of which is a mirror image of the other
• These 2 options are mirror images of each other, but not superimposable
• All amino acids found in proteins are of the L-form
• Glycine (Gly) has no side chain (only an H atom) and is therefore the only non-chiral amino acid
• Humans use one type of chirality

Question 2

Recall amino acids with non-polar and polar side chains

Answer 2

The chemical properties have effects on the protein e.g. its is hydrophobic, so protein folds a certain way

Starred ones are amino acids Dr Pease recommends to learn

1. Glycine – R group is hydrogen (no chorality)
2. Cysteine – forms disuphide bridges
3. Glutamic acid (hydrophilic)
4. Valine (hydrophobic)

Why are these 2 important?

• Sickle cell disease

What happens in sickle cell disease?

• There is a change from Glutamic acid to valine at position 6

Question 3

Recall amino acids and how they change according to pH

Answer 3

Amino Acids with Charged side-chains:

• Arginine (Arg) and Lysine (Lys) at physiological pH are always protonated and therefore basic.
• Histidine (His) is protonated below pH 6.0
• Similarly Glutamic acid (Glu, E) and Aspartic acid (Asp, D) at physiological pH are always negatively charged due to proton donation.

Question 4

Concepts: summarise the concepts of primary, secondary, tertiary and quaternary protein structure

Answer 4

Primary structure

Simply the linear sequence of amino acids that make up the protein.

Standard nomenclature dictates that we write a protein sequence from the amino terminus to the carboxyl terminus.​

Secondary structure

Defined as local structural motifs within a protein, e.g. a-helices and b-pleated sheets.

Their existence within a protein is dictated by the primary structure or amino acid sequence.

The α-helical content of proteins ranges widely, from none to almost 100%.

example: about 75% of the residues in ferritin, a protein that helps store iron, are in α helices.

Tertiary Structure:

Defined as the arrangement of the secondary structure motifs into compact globular structures called domains

Quaternary Structure

Defined as the three dimensional structure of a multimeric protein composed of several subunits.

Oxyhaemoglobin is comprised of 4 subunits (2α and 2β chains)

Question 5

Primary structure: summarise the reaction by which amino acids are joined together, sketch a trimeric peptide and identify the amino terminus, carboxyl terminus and side chains

Answer 5

Peptides are joined by a condensation reaction: photo

Draw a trimeric peptide: like the one in photo + 1 amino acid –> on the right carboxyl terminus, on the left amino terminus

Characteristics of the Peptide Bond (between amino acids)

• There is no free rotation around the peptide bond.
• The C=O and N-H are in the same plane of the molecule.
• The other two bonds in the backbone of the polypeptide are able to rotate. (rotation around the central carbon atom in each amino acid)
• A polypeptide can fold to an overall structure

This has implications for the overall structure of the peptide chain. Only conformations in which side chains do not clash with the main chain (steric hindrance) are allowed.

Steric hindrance – the structure of the molecules are too big to come close enough to react

Question 6

Secondary structure: distinguish between an α-helix and a β-pleated sheet and appreciate the bonds that stabilise their formation

Answer 6

a-Helices:

• Are stabilised by Hydrogen Bonds

Hydrogen Bonds between the C=O of one residue and the N-H of another residue, 4 amino acids along the helix, stabilise the entire structure.

• The side chains of individual amino acids project out from within the a –helix.
• The usage of L-amino acids in proteins means that right-handed helices are favoured.

Proline is a Kinky Amino Acid: When proline is joined to a polypeptide chain, the NH group of the amino acid is lost.

This prevents the side chain from hydrogen bonding with C=O groups of another residue within the helix, thereby distorting the helical conformation, putting a ‘kink’ into it.

b -pleated sheets

• Are stabilized by Hydrogen Bonds

As with the alpha helix, hydrogen bonds between the N-H and C=O groups of two or more b-strands hold the b -pleated sheet sheet together.

• The NH and C=O groups point out at right angles to the line of the backbone. This almost two dimensional sheet is pleated
• Anti-parallel b-pleated sheet OR Parallel b-pleated sheet

Question 7

Tertiary structure: differentiate between the different types of bonds that combine to stabilise a particular protein conformation

Answer 7

What holds a protein together?

1. Covalent bonds (in which two atoms share electrons) are the strongest bonds within a protein and exist in the primary structure itself.

• Covalent bonds can also exist as disulphide bridges. These occur when cysteine side chains within a protein are oxidised resulting in a covalent link between the two amino acids.
• (Disulphide bridge links 2 domains of the chemokine eotaxin)

2. Hydrogen Bonds

• Occur when two atoms bearing partial negative charges share a partially positively charged hydrogen, the atoms are engaged in a hydrogen bond (H-bond).
• Occur either between atoms on different sidechains and the backbone of the protein or between water molecules.

3. Ionic interactions a.k.a. salt bridges

• Arise from the electrostatic attraction between charged side chains e.g. Glu, Asp, Lys and Arg.
• Relatively strong bonds, particularly when the ion pairs are within the protein interior and excluded from water.
• Majority of charged groups are at the surface of the folded protein. There, they can be neutralised by counterions such as salts.

4. Van der Waals Forces

• Transient, weak electrostatic attractions between two atoms, due to the fluctuating electron cloud surrounding each atom which has a temporary electric dipole.
• The transient dipole in one atom can induce a complementary dipole in another atom, with weak attractive properties.
• Alternatively, if the two electron clouds of adjacent atoms are too close, repulsive forces come into play because of the negatively-charged electrons.

5. Hydrophobic Interactions

• Hydrophobic interactions are a major force driving the folding of proteins into their correct conformation.
• Proteins juxtapose hydrophobic sidechains by packing them into the interior of the protein.
• This creates a hydrophobic core and a hydrophillic surface to the majority of proteins.

Folding of Proteins:

• Proteins generally fold into a single conformation of lowest energy.
• This can occur spontaneous or involve other molecules known as chaperones, which bind to the partly folded polypeptide chain and ensure that the folding continues along the most energetically favourable pathway.
• By breaking the bonds that hold the protein together, we can denature the protein into the original flexible polypeptide.
• Common denaturants used within the laboratory are urea (breaks hydrogen bonds) and 2-mercaptoethanol (breaks disulphide bonds).

Question 8

Protein analysis: explain the principles of electrophoresis, and why proteins with single mutations may be separated by electrophoresis depending on the individual mutations; explain the effects that post-translational modifications may have on protein analysis

Answer 8

Amino Acids with Charged side-chains

The state of ionisation of an amino acid provides vital biological properties to many proteins and enzymes, and for this reason cells cannot generally tolerate wide changes in pH

Using techniques such as electrophoresis to separate proteins on the basis of their charge, we can distinguish between normal and mutant forms of proteins which have gained or lost charge e.g. HbA vs HbS

Post Translational Modification of Proteins

Post-translational modifications of proteins add yet more diversity to protein structure.​

N-linked glycosylation, the addition of sugar groups to asparagine (N) residues of the LHR ensures that it adopts the correct conformation in the cell membrane.

Mutation of two asparagines (N) to glutamine (Q) can be picked up by electrophoresis as a reduction in the molecular weight of the LHR.