4 Protein structure and function 2

Question 1

Describe and explain Quarternary Structure

Answer 1

Most cellular proteins are oligomeric (>1 polypeptide chain)

• if an oligomer contains identical polypeptides, it is a homo-oligomer
• if an oligomer contains non-identical polypeptides, it is a hetero-oligomer

A protomer is the smallest distinct unit in a complex (e.g. haemoglobin is a dimer of aß protomer)

Question 2

Describe inter-protein interactions

Answer 2

Subunits normally interact non-covalently (e.g. disulphide bonds)

Protein-protein interfaces:
- contain closely packed non-polar side chains and H-bonds (both main-chain and sidechain)
- are less hydrophilic than solvent-facing protein surfaces, but more hydrophilic than protein interiors
> lower interface-hydrophobicity is associated with transient oligomers
- certain residues found more frequently than at the surface (Arg, Trp, Tyr)
> buried salt-bridges increase oligomerisation specificity
- protein-protein interaction is a pattern-recognition process

Question 3

Describe spatial arrangements in terms of proteins and polypeptide subunits

Answer 3

Polypeptide subunits in oligomers associate with a specific geometry (quarternary)

• in many cases, subunits are symmertrically arranged
• they can show rotational symmetry

Question 4

Describe 2 reasons why proteins form complexes

Answer 4

1. Efficiency/ease of construction
   - can expand function
   - easier repair (and control)
   - can assemble at selected site (s)
   - can reduce genome size (can use subints in more than one complex)
2. Oligomers can make better enzymes
   - size increase can stabilise individual subints
   - multiple binding/active sites
   - subunits permit regulation and cooperativity

Question 5

Describe the 3 main protein types (briefly)

Answer 5

1. Fibrous
   - normally display a single secondary structure
   - e.g. a-keratin from wool + hair
2. Globular
   - exhibit secondary structure elements inter-spaced with loops devoid of regular structure
   - e.g. most cytoplasmic proteins
3. Membrane
   - integral membrane proteins contain one or more hydrophobic domains that span the lipid bilayer

Question 6

Describe globular proteins (using Haemoglobin as an example)

Answer 6

Haemoglobin: The oxygen carrier of blood

• 4 globin subunits (i.e. tetrameric protein)
• Adult form (HbA) contains two alpha (a) and two beta (ß) subunits: a2ß2
• Foetal form (HbF) contain two alpha (a) and two gamma (y) subunits: a2y2

The single haem group per subunit binds O2
- Contains protoporphyrin and iron (II) (Fe2+)

Question 7

Describe the relationship between cooperativity and O2 affinity in haemoglobin

Answer 7

• deoxy-Hb aß dimers are held together by salt bridges and H-bonds
• these weaken when oxygen binds
• the two forms of T (taut) and R (relaxed) respectively

Question 8

Describe haemoglobin, and how it works to bind with oxygen cooperatively

Answer 8

Haemoglobin must bind efficiently in the lungs and release oxygen to tissues
[can’t do this with constant oxygen affinity]

• Instead, Hb binds oxygen cooperatively
  > binding of one oxygen makes the affinity at the other sites higher
  > release of an oxygen reduces the affinity at the other sites

A sigmoidal surve is the signature of cooperativity

Question 9

Describe how a structure of a protein can suggest the location where it functions

Answer 9

Secreted proteins, like Insulin

• contain an N-terminal leader or Signal peptide that interacts with the export machinery in the cell
• it can be exported + inserted into the membrane
• this signal peptide is recognised and directed to rough ER (different acidic/basic residue)

Membrane proteins with external N-termini also have signal peptides
- and they have hyrophobic regions (around 20aa) called transmembrane domains that span the membrane

Question 10

Describe protein modifications that can occur to proteins

Answer 10

Proteins in eukaryotes often undergo covalent modification, usually following ribosomal synthesis (termed Post Translational Modification - PTM):
- these changes occur at specific sequences
- they can regulate protein activity:
> reversible changes include phosphorylation, methylation, and acylation
> irreversible changes can include cleavage
- they can regulate destination:
> e.g. lipidation and glycosylation
> the effect depends on the context (e.g. ubiquitination)

Question 11

Describe glycosylation as a Post-translational modification that can be done to proteins

Answer 11

Glycosylation - attachment of carbohydrates
(common PTM)
- found particularly in extracellular + cell-surface proteins (protects against digestion)
- sugars attached to asparagine (N), serine (S), and threonine (T) residues

It can form two types of proteins:

• Glycoprotein: where carbohydrate is attached as a minor component
• Proteoglycan: where carbohydrate is attached as a major component

Glycosylation has a number of roles:

• aids protein folding and can influence targeting of newly synthesised protein to its destination
• glycosylation is involved in cell-to-cell recognition

Two types of glycosylation can occur:

• N-linked (occurs at N-X sequence) in residue
• O-linked (occurs at O-H of a -OH group)

Question 12

Give an example of glycosylation in a protein

Answer 12

Glycosylation attachments determine blood group

ABO blood groups antigens are O-linked oligosaccharides on RBC proteins and lipids:
- all antigens share the O carbohydrate foundation
- A + B have an extra monosaccharide:
> A: N-acetylgalactaminose
> B: galactose

Question 13

Describe hydroxylation as a Post-translational modification that can be done to proteins

Answer 13

Hydroxylations is the addition of an -OH in proline or lysine

• collagen is a fibrous protein of connective tissue
• collagen polypeptides consist of several hundred repeats of (Gly-Pro-OH-Pro)

Hydroxylated lysine residues are involved in collagen cross-linking

Question 14

Give an example of glycosylation in a protein

e.g. Vitamin C

Answer 14

Vitamin C is required for Pro and Lys hydroxylation

• deficiency leads to scurvy (minor haemorrhages, fatigue, eventual heart failure)
• humans (and other primates) have lost the last enzyme within the ascorbic acid synthesis pathway

Question 15

Describe genetic mutations, and how they can affect protein synthesis

Answer 15

Evolution has optimised proteins for their roles
- Mutations causing amino sequence changes are therefore often deleterious

NB
- if a protein is absent or dysfunctional, the other allele may produce sufficient protein for the effect to be a recessive trait (IF heterozygous)

Question 16

List some effects of genetic mutations

Answer 16

• Disruption to a metabolic pathway
• Impairment or loss of defence against infection
• Protein aggregation
• Dysfunction of a regulatory protein or receptor

Question 17

Describe disruption to a metabolic pathway as a side effect of genetic mutations

Answer 17

Disruption to a metabolic pathway

• loss of products(s) and accumulation of precursor
• e.g. The hereditary disease phenylketonuria, resulting from the loss of the phenylalanine hydroxylase enzyme

Question 18

Describe impairment or loss of defence against infection as an effect of genetic mutations

Answer 18

Mutations within the innate or adaptive immune system

- can compromise their effectiveness/lead to inappropriate activation

Question 19

Describe protein aggregation as an effect of genetic mutations

Answer 19

Protein aggregation

- can lead to disease such as Alzheimer’s and Huntingdon’s

Question 20

Describe sickle cell anaemia as an example of genetic mutation

Answer 20

The mutation present is a single change in a base in DNA - a single change in an amino acid - which changes the B subunit in haemoglobin

• if 1 or the mutated Bs allele is present = asymptomatic
• if both mutated BsBs alleles are present = symptomatic

These mutated sickle haemoglobin molecules associate to form fibres under low O2 levels

These distort the erythrocytes into sickle shapes, which causes them to blocks capillary flow

Question 21

Describe the relation of folding of proteins, and how mistakes can lead to disease

Answer 21

A protein’s function depends on it having a specific 3D structure

• this can be lost during a protein’s ‘lifecycle’
• problems can arise during protein folding (which has to occur rapidly in a highly concentrated environment)

Cells have developed extensive mechanisms to prevent or remove misfolded proteins, e.g.

• chaperones - to mediate folding
• proteasome (via ubiquitin) and lysosome (in autophagy) to mediate breakdown

Clues to a protein’s folded state may come from the exposure of hydrophobic residues

Question 22

Describe protein misfolding disorders (PMDs)

Answer 22

Despite these control measures, proteins can fold into non-native ‘conformers’

• above a certain threshold, these act as seeds for aggregation
• leads to protein-misfolding disorders (50 examples, including Alzheimer’s, Parkinson’s, and Huntington’s diseases)
• they don’t just occur in the CNS (e.g. amyloidosis in liver, spleen and PNS)
• cytotoxic effects come from hydrophobic regions interacting with cellular components like lipids
• many of these disorders appear to be age-related (also genetic factors)