L1: Amino acids and proteins

What are the 4 different groups covalently bonded to the a-carbon of an amino acid?

1. Amino group (-NH2)

2. Carboxyl group (-COOH)

3. Radical group (R-)

4. Hydrogen atom (H-)

State the 3 letter sequence and 1 letter code for each amino acid

• Methionine (Met) [M]

• Valine (Val) [V]

• Alanine (Ala) [A]

• Glycine (Gly) [G]

• Leucine (Leu) [L]

• Serine (Ser) [S]

• Threonine (Thr) [T]

• Isoleucine (Ile) [I]

• Cysteine (Cys) [C]

• Lysine (Lys) [K]

• Arginine (Arg) [R]

• Asparagine (Asn) [N]

• Glutamine (Gln) [Q]

• Aspartate (Asp) [D]

• Glutamate (Glu) [E]

• Proline (Pro) [P]

• Phenylalanine (Phe) [F]

• Tyrosine (Tyr) [Y]

• Tryptophan (Trp) [W]

• Histidine (His) [H]

Which is the only amino acid without a chiral carbon?

Glycine

What is physiological pH?

7.4

What are the ways to classify amino acids?

Based on side chains:

1. non-polar aliphatic side chains

2. aromatic side chains

3. sulfur-containing side chains

4. side chain with hydroxyl (OH) group

5. charged basic side chains

6. charged acidic side chains + derivatives

7. side chain with amide groups

Describe non-polar aliphatic side chains. State which amino acids have them (GLaciers in ALAska VALiantly Locate ISOlated Prowlers)

• Consists of H and C

• Hydrophobic, non-polar (uncharged)

• Chemically non reactive

  • R groups only contain inert methylene (CH₂) or methyl (CH₃) groups

• Longer hydrocarbon chain = Amino acid is more hydrophobic

Solely aliphatic: Glycine, Alanine, Valine, Leucine, Isoleucine

Aliphatic + others: Proline

State which amino acid has an aliphatic cyclic group. Explain its how it affects the structure of the amino acid

Proline

• Proline has its aliphatic side chain bonded back onto the α-amino group and does not fit nicely into any class because it is cyclic.  

• Proline is conformationally rigid. This rigidity of the ring plays a critical role in protein structure. Proline is usually found at bends in the polypeptide

Describe aromatic side chains. State which amino acids have them

• Hydrophobic ring

• Strongly absorb UV light at 280nm

• R-group consists of a phenyl ring, which is responsible for most of the UV properties of proteins

Phenylalanine, Tyrosine, Tryptophan

Describe sulfur-containing side chains. State which amino acids have them

• Has sulfur atom in structure

• 2 Cysteine molecules can form disulfide bond (S-S) from sulfur group

Cysteine, Methionine

Describe side chains with hydroxyl groups (alcohols). State which amino acids have them

• OH side chain can participate in the formation of hydrogen bonds

• Amino acids with hydroxyl groups are important in Phosphorylation reactions

  Phosphorylation → When a phosphate group is added to the O on a hydroxyl group. A H is loss. Phosphorylation reactions are catalysed by protein kinases

Serine, Threonine, Tyrosine

Describe charged basic side chains (-NH₂ and =NH), basic amino acids. State which amino acids have them. (HLA)

At neutral pH, amino group side chains are protonated → Positively charged

Histidine, Lysine, Arginine

Describe charged acidic side chains (-COO^-) Acidic amino acids. State which amino acids have them

At neutral pH, carboxyl group side chains are not protonated → Negatively charged

Glutamate, Aspartate

Describe amide side chains (-CONH₂). State which amino acids have them

Amide side chains (-CONH₂) are uncharged, but can form Hydrogen bonds

Glutamate, Aspartate

What is the difference between essential and non-essential amino acids?

Essential: Cannot be synthesised in the body, has to be obtained from diet

Non-essential: Can be synthesised in the body

Which amino acids are essential (LAMP LIT HTV) and non-essential (AAACGGGPST)?

Essential	Non-essential
LAMP LIT HTV - Lysine - Arginine* - Methionine - Phenylalanine - Leucine - Isoleucine - Threonine - Histidine - Tryptophan - Valine	AAACGGGPST - Alanine - Aspartate - Asparagine - Cysteine - Glutamine - Glutamate - Glycine - Proline - Serine - Tyrosine

Arginine → Because mammals cannot synthesise enough arginine to meet the metabolic needs of infants and children, it is classified as an essential amino acids

What are the properties of amino acids?

At physiological pH (pH 7.4), amino acids are dipolar ions (always charged)

• Amino group is protonated (-NH₃⁺)

• Carboxyl group is unprotonated (-COO^-)

What are the 7 ionisable amino acids? (Triprotic amino acids) LATCHAG

Triprotic → Contains 3 groups that can be ionized (Amino group, carboxyl group, side chain)

Lysine (NH2), Arginine (NH), Tyrosine (OH), Cysteine (SH), Histidine (NH), Aspartic acid (COOH), Glutamic acid (COOH)

What is the charge of an ionizable group on an amino acid dependent on?

The charge of an ionizable group on an amino acid is dependent on the pKa of the group

What is pKa?

pKa → pH at which concentrations of protonated and unprotonated forms are equal.

pKa = -log(Ka)

• pKa <2 means strong acid

• pKa >2 but <7 means weak acid

• pKa >7 but <10 means weak base

• pKa >10 means strong base

Does protonation/deprotonation occur at low/high pH?

How to determine charge on an ionisable group

To determine the charge on an ionizable group, we need

1. pKa of ionizable group

2. pH

State how many H+ ions there are in the solution and the charge when,

pH > pKa

pH < pKa

pH > pKa means have very little H+ ions (protons) in the solution

pH < pKa means have a lot of H+ ions (protons) in the solution

pH > pKa -- negative charge (if have)

pH < pKa -- positive charge (if have)

What is the isoelectric point (pI)?

Isoelectric point (pI) → pH at which overall charge of molecule = 0

The overall charge of a protein or amino acid is dependent on the pI

Same logic as pKa (E.g pH > pKa: 0 or -. pH > pI: -)

pH > pI → -

pH < pI →  +

pH = pI →   0

What is the formula for calculating the Isoelectric point for diprotic amino acids?

pI = (pKa_C + pKa_N )/2

How to calculate isoelectric point for triprotic amino acids?

To determine pI, need to perform a titration

At low pH, ______ protons are present, ______ the chance of protonation.

At high pH, ______ protons are present, ______ the chance of deprotonation

At low pH, more protons are present, increasing the chance of protonation

At high pH, less protons are present, increasing the chance chance of deprotonation

What are the functions of proteins? (DESCC) Give examples.

1. Defense / immunity

   • Immunoglobins, antibodies

2. Energy production 

   • ATP

3. Structure/ Mechanical function

   • Collagen, keratin

4. Catalyst

   • Enzymes 

5. Contraction/movement

   • Actin, myosin, tubulin

What is the N terminus and C terminus?

 

N terminus: free amino end of a peptide

C terminus: free carboxyl end of a peptide. Where more amino acids are added on.

All proteins have free amino group at N terminus and free carboxyl group at C terminus.

What type of reaction is peptide bond formation?

Condensation reaction

How do amino acids form proteins?

Carboxyl group of amino acid A reacts with amino group of amino acid B, forming a peptide bond, releasing one molecule of water.

What is a Dipeptide, Tripeptide, Polypeptide?

Dipeptide → two amino acids joined together

Tripeptide → three amino acids joined together

Polypeptide → many amino acids joined together

What are the types of protein structure?

1. Globular

   • Hydrophobic interior, hydrophilic exterior

   • e.g. enzymes, carrier proteins, regulatory proteins

2. Fibrous

   • Provide mechanical support

   • e.g. collagen, keratin

What are the 4 levels of protein structure?

1. Primary structure

2. Secondary structure

3. Tertiary structure

4. Quaternary structure

Describe primary structure. Give examples

• Sequence and number of amino acids held together by peptide bonds in a polypeptide chain

• E.g Ala-Arg-Asp-Gly

Describe secondary structure (alpha helix)

1. Alpha-helix

• Regular, repeating motif in protein

• In globular, water-soluble proteins

• Composed of right-handed spiral amino acid chain stabilised by H bonds parallel to helix axis

In an alpha-helix, the carboxyl group of residue n forms H bond with NH group of residue n +4 

Describe secondary structure (beta pleated sheets)

2. Beta-pleated sheets

• Regular, repeating motif in protein

• In rigid, insoluble proteins

• Made of polypeptide chains running parallel or anti-parallel to one another

• Stabilized by H bonds formed perpendicular between adjacent chains

Describe secondary structure (Loops and turns)

3. Loops and turns

• Cause directional change in the polypeptide backbone

• About 5 a.a

Describe tertiary structure by stating the bonds.

Protein folding (bonds)

Disulfide bonds (-S-S-): Between two Cysteine amino acids, Strong covalent bonds

Hydrogen bonds: Between O and H, weak bond

Ionic electrostatic bonds: Between COO^- and -NH₃⁺, weak bond

Hydrophobic/Hydrophilic interactions: Between R groups, weak bond

Explain what is a domain in tertiary structure

• A structurally independent region of polypeptide that folds independently of other domains

• Have specific function

• Can be removed & added to other proteins by genetic engineering

• Proteins can have 1 to many domains depending on protein size

Describe quaternary structure

• Multi-subunit proteins - composed of several protein subunits grouped together.

• E.g Hemoglobin (Consists of four tightly packed chains: Two alpha chains, Two beta chains)

What is protein folding dependent on?

1. Primary structure

2. Bonds

   • Disulfide, Hydrogen, Ionic (electrovalent), Hydrophobic / Hydrophilic interactions

   • Polar vs. Non-polar R groups

3. Chaperone proteins (heat-shock proteins)

What are chaperone proteins?

• Large multi-subunit protein

• Facilitate folding of newly synthesised proteins to prevent incorrect folding

• Over-produced when cells exposed to heat to prevent protein denaturation

Explain Post translational modification

Post translational modification → When newly synthesized polypeptide chains are modified immediately after translation

• Post translational modifications are critical for the functional capability of the protein

What are the types of post translational modification of proteins

1. Remove part of amino acid chain

   • N terminal or C terminal residue

   • Part of polypeptide (e.g. insulin)

   • Signal sequence

2. Remove/add functional group

   • Add carbohydrate group

   • Form complex with metal ion

   • Phosphorylation / dephosphorylation

Insulin is produced by ____ to ____. When is insulin secreted? What are the effects of insulin?

• Produced by pancreas to control blood sugar levels

• Secreted when blood glucose levels are high

Has the following effects:

• Increases glucose uptake into muscle & fat cells

• Stimulates glycogen synthesis

Describe the post translational modification for insulin

Removal of part of a polypeptide sequences (E.g Post translational modification for Insulin)

1. Signal sequence removed from preproinsulin, producing proinsulin

2. Proinsulin undergoes further modifications, where a peptide sequence within proinsulin is removed

3. This leaves behind 2 fragments linked by a disulfide bond, thus forming insulin

Briefly describe and give an example of the post translational modification process of attaching carbohydrate groups

Carbohydrate + polypeptide = glycoproteins

E.g Blood ABO antigens

Briefly describe and give an example of the post translational modification process of complexing with metal ions

Haemoglobin made of 4 polypeptide chain forming 4 heme subunits. 4 iron atoms required for each heme

Briefly describe the post translational modification process of phosphorylation and dephosphorylation

Kinases → enzymes that phosphorylate proteins

Phosphatases → enzymes that remove phosphate from protein

Phosphorylation: Uses ATP to add phosphate to protein (ATP → ADP)

Dephosphorylation: Phosphatase removes phosphate group