Week 8 (Proteins II)

Question 1

What is the tertiary structure?

Answer 1

The tertiary structure of a protein is the overall 3-dimensional arrangement of all atoms in a polypeptide chain.

• The way in which the α-helices and β-sheets fold up together to form a compact structure.
• Amino acids which are far apart in the amino acid sequence may end up close to each other in the final folded structure.
• Stabilised by multiple weak interactions.

Question 2

What is the main factor in driving folded structure?

Answer 2

• The main factor in driving folded structure is the hydrophobic effect.
• Most proteins are in aqueous environment.
• Hydrophilic side chains stick out into the water.
• Hydrophobic side chains bury in the core away from the water.

Question 3

What determines protein folding?

Answer 3

Proteins fold into the lowest energy conformation (shape)

Question 4

Describe Non-covalent stabilising interactions

Answer 4

• Van der Waals between hydropjobic side chains
• Ionic bonds (electrostatic attraction) between oppositely charged side chains
• Hydrogen bonds between polar side chains

Question 5

What are prosthetic groups?

Answer 5

A non-protein group forming part of or combined with a protein

• termed prosthetic groups, co-factors or co-enzymes
• Tightly bound into the 3-D structure of the protein
• Can be critical for function
• Examples include haem groups, metal ions, lipids

Question 6

Hydrophobic effect example – horse heart cytochrome C

Answer 6

Hydrophobic residues burried inside the protein core whereas the hydrophilc residues produe outward

Question 7

Hydrophobic effect example – myoglobin

Answer 7

Again, hydrophobic residues burried inside the protein core whereas the hydrophilc residues produe outward

Question 8

What are protein motifs?

Answer 8

A motif or fold is a recognisable folding pattern involving 2 or more secondary structure (supersecondary) elements for example:

• helix loop helix→The motif is characterized by two α-helices connected by a loop
• coiled coiled → in which 2–7 alpha-helices are coiled together like the strands of a rope
• Helical bundle → composed of several alpha helices that are usually nearly parallel or antiparallel to each other
• Beta-alpha-beta (β-α-β) → parallel beta-strands are connected by longer regions of chain which cross the beta-sheet and frequently contain alpha-helical segments
• Beta hairpins → short loop regions between antiparallel hydrogen bonded beta-strands
• Greek key → four adjacent antiparallel strands and their linking loops
• β-meander→ 2 or more consecutive antiparallel β-strands linked together by hairpin loops

Question 9

What kind of protein motifs are there?

Answer 9

• Ranges from very simple motifs such as β-α-β loop to elaborate structures such as the β-barrel
• Can represent only a small part of the protein or the whole structure.
• Several motifs can join together to create larger common folds.

Question 10

What are protein domain?

Answer 10

• A conserved part of a given protein sequence and tertiary structure that can evolve, function, and exist independently of the rest of the protein chain.
• Each domain forms a compact three-dimensional structure and often can be independently stable and folded into a compact structure.

Question 11

What are large single polypeptide proteins that often fold into two or more domains joined together by?

Answer 11

flexible linkers

Question 12

Do different domains may have distinct functions?

Answer 12

Yes

Question 13

Describe the domains of cell surface protein CD4?

Answer 13

Cell surface protein with as 4 similarly folded immunoglobulin (Ig) domains (D1-D4), a transmembrane domain and a cytoplasmic tail domain

Question 14

Src, comprises a C-terminal protein kinase domain. How many regulatory domains does it have?

Answer 14

Two

Question 15

Example protein structures: α + β

Answer 15

α and β regions somewhat segregated

Question 16

Example protein structures: α/β

Answer 16

α and β segments interspersed or alternating

Question 17

What are protein families?

Give an example

Answer 17

• Many proteins can be grouped into families, having similarities in amino acid sequence and structure, yet distinct functions.
• Folded stable proteins with useful properties have been modified during evolution to enable new functions to be carried out.

Elastase and chymotrypsin are members of the serine protease family therefore have very similar structures.

Question 18

What is the Quaternary structure?

Answer 18

The quaternary structure of a protein is when multiple polypeptide chains join together to form multisubunit structures.

• Can range from two polypeptides to hundreds.
• Often termed oligomers.
• Each separate chain is called a subunit.
• Stabilised by the same types of multiple weak interactions as tertiary structure.

Question 19

From dimers to huge multisubunit assemblies

Answer 19

Simplest form of quaternary structure is a dimer.

• Can be homo-oligomers or hetero-oligomers.
• Can often form symmetrical or repeating structures.

Question 20

Describe the quaternary structure of Haemoglobin

Answer 20

A tetramer comprising two α subunits and two β subunits

Question 21

Describe the nature of disulphide bonds

Answer 21

• Cysteine residues have unique chemistry.
• Two cysteines in close proximity will form a disulphide bond.
• Disulphide bonds are covalent cross-links between the sulfhydryl (SH) side chains.
• An S− anion from one sulfhydryl group acts as a nucleophile, attacking the side chain of a second cysteine to create a disulfide bond
• They significantly stabilise protein structure and facilitate folding progression toward the native state by limiting unfolded or improperly folded conformations

Question 22

What are intrachain disulphide bonds?

Answer 22

Between two cysteines in the same polypeptide chain

Question 23

What are interchain disulphide bonds?

Answer 23

Between cysteines in different polypeptide chains

Question 24

Why does Protein denaturation occur?

Answer 24

Protein folded structure held together predominantly by many weak forces

Question 25

What factors can cause protein denaturation?

Answer 25

Heat, extreme pH, detergents, urea or guanidium chloride can all disrupt these interactions and cause denaturation of your protein

Question 26

What can protein denaturation result in?

Answer 26

Results in loss of function – because function is dependent on 3D folded shape

Question 27

What can break disulphide bonds?

Answer 27

Disulphide bonds can be broken using reducing agents such as dithiothreitol or β-mercaptoethanol

Question 28

When you remove the denaturant, can the protein re-fold back into its structure and regain biological activity?

Answer 28

Yes and this is called renaturation

Question 29

Is denaturation always permanent?

Answer 29

No

Question 30

Protein folding

Answer 30

• Polypeptides fold rapidly by a stepwise process.
• Polypeptides fold into their 3D structure whilst still on the ribosome, co-translational.

Question 31

Define co-translational

Answer 31

Polypeptides fold into their 3D structure whilst still on the ribosome, co-translational

Question 32

Amino acid sequence defines structure – genetic disease: Cystic fibrosis

Answer 32

Small mutations in genetic sequence can have quite dramatic effects on the protein produced

One of the most common genetic diseases

• 70% of cases results from deletion of the codon for F508 of the cystic fibrosis protein
• A single amino acid missing form a protein of 1480 amino acids
• Mutation affects the way the protein folds, it isn’t at all stable, and just gets degraded in the ER.
• No functional protein – patient gets ill

Question 33

Amino acid sequence defines structure – genetic disease: Sickle cell anaemia

Answer 33

A single nucleotide mutation (A→T) in the gene encoding Haemoglobin

• Results in substitution of a hydrophilic amino acid (E- Glutamic acid) for a hydrophobic amino acid (V-Valine)
• Causes the haemoglobin proteins to aggregate
• Distorts the shape of red blood cells – they get destroyed more quickly than usual.