Lecture 12-17

Question 1

Describe the general characteristics of structural proteins?

Answer 1

Long, filmentous, insoluble, contain unusual amino acids (through post-transnational modification) and often contain cross-linked polypeptide chains.

Question 2

Where are alpha keratins found?

Answer 2

Outermost layer of the skin, horns, hooves and hair.

Question 3

Where are beta keratins found?

Answer 3

Feathers, scales.

Question 4

What is the typical MW of intermediate filaments of keratin?

Answer 4

40-60,000 or 50-70,000.

Question 5

Describe the structure of alpha keratin?

Answer 5

Polypeptide chain forms a right handed alpha helix. Two of these polypeptide chains twist together to form a left handed helix, known as a coiled coil. Each turn 3.5 residues with a pitch of 0.51. Coiled coil ~45 nm long. Coiled coils dimerise to form a protofilament. Two protofilaments supercoil to form a righthanded protofibril. Eight protofibrils form a microfibril.

Question 6

What are the common amino acids in keratin?

Answer 6

Alanine, leucine, arginine and cysteine.

Question 7

Which amino acid in keratin forms disulphide bonds between coiled coils?

Answer 7

Cysteine.

Question 8

Profilaments in keratin are stabilised by…

Answer 8

…hydrophobic interactions.

Question 9

Keratin is resistant to digestion by…

Answer 9

…proteases pepsin or trypsin, insoluble in dilute acids, alkalines, water and organic solvents.

Question 10

Keratins are extracted using…

Answer 10

…reducing agents such as thioglycollate, dithiothreitol or mercaptoethanol, which cleave disulphide bonds.

Question 11

How are types of keratin distinguished?

Answer 11

Isoelectric points. E.g. Type 1 is acidic pI 4.9-5.4. Type 2 is basic pI 6.5-8.5 pH.

Question 12

What post translational modification is most important in keratins?

Answer 12

Phosphorylation and dephosphorylation. Affect the equilibrium of soluble keratins. Phosphorylation of amino acids in the head domain changes the overall net charge of this region thereby preventing the assembly of adjacent keratin filaments.

Question 13

Where are the two types of collagen found in the body?

Answer 13

Type 1: tendons, ligaments, bones.

Types 2: cartilage.

Question 14

What is the amino acid composition of collagen?

Answer 14

33% Gly, 11% Ala, 10% Pro, 5% Hyp, 0.6% Hyl.

Question 15

What characteristic motif is associated with collagen?

Answer 15

Gly-Pro-Ala-Gly-Pro-Ala. Every third residue being Gly is essential for structure.

Question 16

What is the structure of collagen?

Answer 16

Three chains, each ~1000 amino acid residues, intertwined in a right-handed not a helix. Length 300 nm, diameter 1.4 nm, MW 285 kDa. Tropocollagen. These tropocollagen strands are aligned in a staggared arrangement, 64 nm staggering with a start to end gap between strands of 40 nm.

Question 17

Why does collagen contain alot of proline?

Answer 17

To prevent the formation of too many hydrogen bonds that would hinder flexibility. Though some hydrogen bonds are required for stability. Modified proline, hydroxyproline serves as a hydrogen bond acceptor and raises the melting temp of collagen above body temp.

Question 18

Why is it important that every thrid residue in collagen in glycine?

Answer 18

Line the interior of the twisted chains, their lack of side chains allow for tight packing. Other residues bulky sides chains are orientated outwards.

Question 19

Why does collagen contain alot of lysine?

Answer 19

Lysine side chains are converted aldehydes, allysine by lysyl oxidase. Allysine side chains interact with other allysine side chains via allysine aldol condensation forming cross links.

Question 20

Where is elastin found?

Answer 20

Arterial walls and ligaments.

Question 21

What is the amino acid composition of elastin?

Answer 21

33% Gly, 22% Ala, 14% Val, 11% Pro, 7% Leu and also Lys.

Question 22

Describe the structure of elastin?

Answer 22

Lacks secondary structure, random coil, 95% hydrophobic residues, and 74 kDa. Contain desmosine and isodesmosine nodes. Tropoelastin forms from the polymerisation of monomers.

Question 23

Where is fibrillin found in the body?

Answer 23

Microfibrils are found in the skin, blood vessels, perichondrium and tendons. Normally alongside elastin.

Question 24

Fibrillin contains repeat motifs…

Answer 24

… of epidermal growth factor like modules. 47 EGF motifs of which 43 bind to Ca2+. Ca2+ binding regulates flexibility.

Question 25

What is the amino acid composition of silk fibroin?

Answer 25

45% Gly, 30% Ala, 12% Ser, and 5% Try and some Pro.

Question 26

Describe the structure of silk fibrion?

Answer 26

Anti-parallel pleated beta sheets stabilised by hydrogen bonds.

Question 27

How many nucleotide pairs in length are cis-regulatory sequences?

Answer 27

5-10

Question 28

What is the characteristic TATA box sequence?

Answer 28

5’-TATAAA-3’

Question 29

How many nucleotides separate the gene and promotor region in eukaryotes?

Answer 29

-75 to -25

Question 30

Describe the mechanism of transcriptional regualtion?

Answer 30

Transcription factor II binds to TATA. Specific TF binds to the enhancer region. TFII and specific TF come into contact via DNA bending. Recruitment of RNA Pol II.

Question 31

Describe the structure of TFII and how it binds to the TATA box region of the DNA?

Answer 31

Has a highly conserved C terminus. Beta sheet with a leading hairpins which line with the interface of the DNA. The helices on the outside are structural which allow the correct alignment of the beta sheets. Binding of the transcription factor occurs in the minor groove of the DNA. Binding causes DNA to bend 100 degrees, opening the DNA by breaking the H bonds, local unwinding for interaction with RNA Pol II.

Question 32

Describe the structure and binding of helix-turn-helix motif?

Answer 32

The pair of helix-turn-helix motifs are are symmetrically arranged. The size of the HTH matches the size of the major groove ~12 A. Specificity of HTH interaction enables specific H bonding between exposed side chains in the major groove. Stabilised by non-polar interactions. Tow helices insert into major groove acting as recognition helices.

Question 33

Name the groups in which helix-turn-helix can be classified?

Answer 33

Dihelical, trihelical, tretra-helical and winged HTH

Question 34

Describe the structure of zinc fingers?

Answer 34

Conical two cystines and two histidines. Fit into the major groove and contains a recognition alpha helix.

Question 35

Describe the structure of a leucine zipper motif?

Answer 35

Consists of of an alpha helical coiled coil (heptad organisation) a pair of DNA binding domains. Leucine is present at N and N+7 over 35 residues. Leucine zippers form dimers held together by a coiled coil.

Question 36

Describe the structure of a homeodomain?

Answer 36

Consists of three helices, 1 and 2 are antiparallel, helix 3 is perpendicular to both and is the recognition helix that is embedded in the major groove. The hydrophobic face of helix 3 packs against helices 1 and 2 to form the interior of the protein. The N terminal domain contains a highly conserved recognition motif RGR which binds to the minor groove.

Question 37

Define a RNA binding protein?

Answer 37

Proteins that bind to the double or single stranded RNA and participate in forming ribonucleoprotein complexes. exist in cytosol and nucleus.

Question 38

Protein synthesis follows RNA from…

Answer 38

5’ to 3’ direction.

Question 39

Name three methods of studying protein RNA interaction?

Answer 39

Coprecipitation of RNA with specific RNA-binding protein, polyadenylated RNA-binding protein, and orthoganol organic phase seperation.

Question 40

What % of the proteosome is membrane proteins?

Answer 40

20%

Question 41

Give an example of a membrane protein drug target?

Answer 41

Beta adrenergic receptors which regulate muscle relaxation. Agonists are used to treat asthma.

Question 42

What is the role of cystic fibrosis transmembrane conductance regulator?

Answer 42

Involved in maintaining nice fluid mucus secretion in the lungs and intestine. Common mutation resulting in the loss of phenylalanine 508 causes cystic fibrosis.

Question 43

Name the six functions a membrane protein can have?

Answer 43

Receptor, transporter, channels, structural, enzyme and marker.

Question 44

Describe an integral membrane spanning protein?

Answer 44

Hydrophobic surface: leu, phe, trp, ile, val to enable interaction with the lipids of the membrane. Majority have a hydrophobic core sometimes hydrophilic.

Question 45

Describe a peripheral membrane protein?

Answer 45

Water soluble proteins attached to the membrane, therefore has a hydrophilic surface (asp, glu, ser, thr) and hydrophobic core.

Question 46

Describe the structure of a membrane protein composed of alpha helices bundles?

Answer 46

All the polar groups within the peptide chain interact to form hydrogen bonds, between 1-5, 2-6, to prevent the exposure of the backbone polar/hydrophilic atoms. 3.6 amino acid turns with side chain orientated outside of the helix. This is useful as polarity is very unfavourable in membrane environments.

Question 47

Describe the structure of a membrane protein composed of a beta sheet?

Answer 47

Beta sheet forms a barrel, forms non-specific pores, substrate flux down diffusion gradient. Hydrophobic residues are orientated to face outwards of the barrel and hydrophilic inwards. These porins have stabilised trimeric interactions. Pore size is controlled by the number of strands, at 8-22 strands typically even number stabilised by circular peptide bond formation.

Question 48

Aquaporins are not…

Answer 48

…beta barrels.

Question 49

How can alpha helices be predicted in membrane proteins?

Answer 49

Hydrophobicity profiling. Look for peaks of 20-25 amino acids as need about 25 amino acid in an alpha helix to span a membrane.