1. Protein evolution

Question 1

Name 3 mechanisms of new gene acquisition

Answer 1

Horizontal gene transfer, de novo origination and duplication-divergence

Question 2

Define horizontal gene transfer as a means for new gene acquisition

Answer 2

A gene from a different organism is inserted into the genome via recombination or transposition

Question 3

Define de novo origination as a means for new gene acquisition

Answer 3

Existing genes spontaneously mutate to diverge and gain a new function

Question 4

Define duplication-divergence as a means for new gene acquisition

Answer 4

An existing gene duplicates and acquires new functions through mutation, deletion and insertion, creating a protein paralog

Question 5

Name two ways in which multi-domain eukaryotic proteins can arise

Answer 5

1) Alternative splicing 2) Exon shuffling

Question 6

Name 4 aspects that can differ between isoforms of a protein

Answer 6

Function, efficiency, specificity, mechanism of action

Question 7

Name 3 examples of slowly evolving proteins

Answer 7

Histones, cytochrome, insulin

Question 8

Name an example of a quickly evolving protein

Answer 8

Immunoglobulin G

Question 9

Define divergent evolution

Answer 9

A protein and its ortholog develop into distinct functions

Question 10

Name two features of divergently evolved proteins

Answer 10

Similar tertiary structure, related but not identical functions

Question 11

Name an example of a divergently evolved protein domain, its function and 4 proteins where this domain is found

Answer 11

RNAse H-like catalytic domain
Cleaves DNA/RNA hybrids or dsDNA, inserts DNA into new genomic sites
Found in bacterial RNAse H, Tn5 bacterial transposase, Mu bacteriophage and RSV integrase

Question 12

Define convergent evolution

Answer 12

Two completely different proteins evolve independently to become analogous

Question 13

Name two features of convergently evolved proteins

Answer 13

Similar/identical functions, different structures and sequences

Question 14

Name an example of a convergently evolved protein domain, its function and mechanism, and 2 proteins where this domain is found

Answer 14

Serine protease
Cleaves peptide bonds using an Asp/His/Ser catalytic triad
Subtilisin (prokaryotic) and chymotrypsin (eukaryotic)

Question 15

Define directed evolution

Answer 15

An unnatural mechanism with a goal to create a protein with a specific function or specificity

Question 16

Name 2 examples of proteins that have undergone directed evolution, how evolution was performed, their functions and applications

Answer 16

1) Variant of subtilisin E; cleaves peptide bonds in organic solvents; has 10 substitutions near the active site that make the surface more hydrophobic, allow internal crosslink and stabilise the active site; used in synthesis of unusual polymers
2) Serine-ligated cytochrome P450; catalyses stereoselective production of highly strained carbocyclic compounds; has a heme that is serine-ligated to ethylenediamine that can catalyse alkyne cyclisation; used in chemical and material synthesis due to high internal energy

Question 17

What is the advantage of repeat expansions?

Answer 17

Simple way to increase complexity of a protein through self-association

Question 18

How do repeat expansions originate?

Answer 18

Via tandem duplication and genetic recombination

Question 19

Name 3 functions of repeat expansions

Answer 19

1) Increase and diversity binding surface area for PPIs
2) Confer new functions
3) Change functions

Question 20

Name 4 examples of structural motifs repeats that enlarge surface binding area for PPIs

Answer 20

1) Leucine-rich repeats
2) Kelch repeats
3) Heat repeats
4) TPR repeats

Question 21

Name 3 structural features of leucene-rich repeats

Answer 21

Each repeat has a hydrophilic a-helix that faces the solution; b-sheet that interacts with neighbouring repeats to form a curved, horseshoe-like shell; and a leucine residue that forms a hydrophobic core

Question 22

Name 2 structural features of Kelch domains

Answer 22

Each domain has 4-5 antiparallel b-strands whose termini interact to form a closed circular structure

Question 23

Describe the structure of HEAT domains and the overall shape of a multidomain structure

Answer 23

Domains are helical-like, form a solenoid structure

Question 24

Describe the primary structure of TPR domains and their role in cells

Answer 24

Composed of 3-16 tandem repeats of a 34 amino acid stretch, forming helical structures. Often found in chaperones where it promotes assembly of multi-protein complexes

Question 25

Name an example of a parental and evolved protein where repeat expansion of a structural motif has resulted in a change of function

Answer 25

Pancreatic trypsinogen evolved into anti-freeze protein via 41x repetition of a small region between within the gene

Question 26

Describe the primary structure of the repeated sequence of anti-freeze protein in arctic/deep seawater fish

Answer 26

Composed of alanine-rich repeats with regularly spaced threonines

Question 27

Describe the secondary structure of the repeated sequence of anti-freeze protein in arctic/deep seawater fish

Answer 27

An a-helix with alanines and evenly spaced threonines on the same face of the helix that is facing ice

Question 28

Describe the tertiary structure of the repeated sequence of anti-freeze proteins

Answer 28

No hydrophobic core, stabilised via H-bonds, disulphide bonds or divalent metal ions (e.g. Ca2+). Have a regularly repeating pattern. Polar and charged amino acids found on the water-facing side

Question 29

Describe the proposed mechanism of ice-binding by anti-freeze proteins

Answer 29

The ice-facing side binds water molecules and arranges them into a crystal-like arrangement that can merge into ice crystals. Anti-freeze protein then causes curvature of the ice surface, making it thermodynamically unfavourable to add more water, halting growth of the ice crystal

Question 30

Name an example of a protein where repeat expansion has resulted in a new function

Answer 30

Bacterial type II topoisomerase

Question 31

Describe the mechanism of type II topoisomerase-mediated DNA supercoiling

Answer 31

1) G segment of DNA binds to GyrA/ParC subunit 2) T segment of DNA is captured by GyrB/ParE subunit 3) 2xATP binds and changes conformation of GyrB/ParE such that T segment is now sealed 4) ATP is hydrolysed by GyrB/ParE which transfers energy to GyrA/ParC to induce a dsDNA break in G segment 5) A conformational change occurs such that both strands of the T segment pass through the dsDNA break in G segment and are released 6) G segment is resealed, released, 2xADP are lost

Question 32

Name 2 functions of bacterial gyrase

Answer 32

1) Creates negative supercoiling | 2) Creates interwound writhes

Question 33

Name 3 functions of topoisomerase IV

Answer 33

1) Relaxes supercoiled DNA 2) Removes writhe 3) Can decatenate two circular plasmids

Question 34

Name two domains of GyrB/ParE homologous proteins

Answer 34

ATPase and Mg2+ binding

Question 35

Name two domains of GyrA/ParC homologous proteins

Answer 35

N-terminal domain (DNA binding and cleavage) and C-terminal domain (DNA binding and regulation)

Question 36

Which domain of bacterial topoisomerase II proteins contains a repeated motif and which proteins is it found in?

Answer 36

The C-terminal domain of GyrA/ParC

Question 37

What is the structure, location and function of C-terminal domain in GyrA/ParC proteins?

Answer 37

Composed of duplicated blade-like motifs that form a bladed pinwheel, located at the outer edge of the GyrA/ParC dimer, interacts with sequences flanking T and G segments

Question 38

What is the main structural difference between GyrA and ParC C-terminal domains?

Answer 38

GyrA CTD contains 6 repeats of the blade-like motif and forms a closed circle. ParC CTD contains 5 repeats of the motif and forms an open pinwheel.

Question 39

What is the functional effect of the difference in number of repeated motifs in the CTD of GyrA/ParC proteins?

Answer 39

In GyrA, the pinwheel is closed, therefore DNA can fully wrap around the domain. This allows gyrase to create writhe between closely spaced G/T segments, e.g. in the same plasmid, to create writhe. In ParC, the pinwheel is open, therefore DNA cannot fully wrap. This restricts the substrate to plasmids where G and T segments are further away to remove writhe, or that belong to 2 different plasmids to decatenate them

Question 40

Define a protein domain and a protein subunit

Answer 40

Domain: an independently folding protein structure OR a distinct evolutionarry conserved amino acid sequence Subunit: an independent polypeptide chain

Question 41

Name two difficulties in structural studies of multi-domain proteins

Answer 41

1) Flexibility of loops and linkers between domains reduces the chance of crystallisation 2) Unstructured regions can promote aggregation instead of crystallisation

Question 42

Name 2 steps in defining domain boundaries computationally

Answer 42

1) Use tools to predict boundaries, secondary structure, flexibility, disorder and coiled-coil regions from sequence 2) Align the sequence to that of homologous proteins

Question 43

What is the main technique used to detect domain boundaries experimentally?

Answer 43

Limited proteolysis

Question 44

Describe the steps of limited proteolysis

Answer 44

1) Digest the native protein with protease(s) while limiting [protease] or time 2) Analyse digested protein by 2a) SDS-PAGE or 2b) MS 3a) Purify digests and sequence the N-terminus to get domain boundary or 3b) determine domain boundaries from mass

Question 45

Name 4 proteases that can be used for limited proteolysis

Answer 45

Glu-C, trypsin, chymotrypsin, pepsin

Question 46

On an SDS-PAGE gel, what would protein fragments with multiple domains vs one single domain look like after limited proteolysis?

Answer 46

Fragments with multiple domains would appear as multiple bands, fragments with a single domain would appear as a single band

Question 47

Name 5 advantages of a multi-domain protein architecture

Answer 47

1) Facilitates evolution of new functions 2) Facilitates regulation and control 3) Can create conformationally-adapted ligand binding sites 4) Simplifies protein folding and assembly 5) Strengthens intramolecular binding

Question 48

Name an example of a multi-domain protein where a ligand binding site only appears after a conformational change. What are its functions, structure, ligands and how is the ligand binding site built?

Answer 48

CHD1 ATPase motor. Chromatin remodelling protein. Composed of two lobes that are in open form when inactive, and a chromodomain that occludes the DNA binding surface. ATP binds to lobe 1 but is not hydrolysed until DNA binds to lobe 2 and induces a conformational change that brings the lobes together. Arginine fingers in lobe 2 can then contact ATP and induce hydrolysis, which gives energy for DNA sliding.

Question 49

What is the folding rate of protein inversely propotional to?

Answer 49

N^3, where N=number of amino acids

Question 50

How does multi-domain architecture simplify protein folding? Name 4 reasons why this is desirable.

Answer 50

Domains are smaller than whole proteins, therefore fold more rapidly. This is desirable to prevent misfolding, proteolysis or unwanted interactions, and is less limited by chaperone cavity size.

Question 51

Name two ways in which multi-domain protein architecture strengthen intramolecular binding

Answer 51

The interdomain linker places the domain in correct orientation with respect to one another and brings them closer together for interaction

Question 52

Name an example of a protein where the interdomain linker has an additional function

Answer 52

X11/Mint family of neuronal proteins that regulate neuronal signalling

Question 53

Describe the domain structure of X11/Mint neuronal proteins

Answer 53

Contain a PTB domain that binds APP, two PDZ domains that are involved in signal transduction, and a C-terminus with conserved YI/YL residues at the end

Question 54

What is the role of conserved YI/YL residues at the C-terminus of X11/Mint neuronal proteins?

Answer 54

Bind to a hydrophobic pocket within PDZ1, which allows C-terminus to wrap around the tandem PDZ domains and occlude the binding site

Question 55

How is X11/Mint autoinhibition released and how this was detected experimentaly?

Answer 55

Phosphorylation of conserved Y residue at -1 from C-terminus disrupts its hydrophobic interaction with PDZ1. This was discovered through Y-1E mutation, which introduces a negative charge in the same way phosphorylation does and also releases autoinhibition.