Lectures 4 - 7

Question 1

Ways to modify protein structure

Answer 1

1. Chemical reagents
2. Unnatural amino acids
3. In vitro/in vivo synthesis

Question 2

Use of chemical reagents to modify protein structure

Answer 2

Add a reagent you expect to react with a chemical group in the protein structure - it is non-selective and gives mixed products

Question 3

Use of unnatural amino acids to modify protein structure

Answer 3

1. Allows a specific function to be added

2. Can expand genetic code through promiscuous translation, suppressor codons and orthologous tRNA/AATS pairs

Question 4

SupE mutation in E.coli

Answer 4

tRNA recognises UAG as a coding codon (pairs with GUC), resulting in a read-through of the STOP codon.

Question 5

Requirements to incorporate unnatural aa in vivo

Answer 5

Unnatural aa
Cognate tRNA (that binds to aa)
Cognate aminoacyl-tRNA synthetase

Question 6

Problems with incorporating unnatural aa in vivo

Answer 6

Costly, low yield
Requires special aa to be acylated - in vitro this can be carried out via chemical charging but in vivo must use modified tRNAs/AATS
Requires orthologous tRNA/AATS pairs e.g. don’t want special tRNA to be charged with normal aa
Requires special codons - use unused/low frequency ones

Question 7

How to engineer tRNA so it is only recognised by cognate AATS

Answer 7

1. Make a mutant tRNA ortholog library
2. Select tRNAs that are recognised and acylated by cognate AATS
3. Kill/remove tRNAs acylated by native AATSs

Question 8

How to engineer AATS to specifically recognise unnatural amino acid and acylate cognate tRNA

Answer 8

1. Produce a library of AATS mutated in the amino acid binding site
2. Live or die selection - kill all AATS that incorporate natural aa

Question 9

In vivo synthesis of unnatural aa

Answer 9

It is more efficient to use host machinery:
1. Start with endogenous precursor
2. Use biosynthetic enzymes
3. Synthesised aa incorporated via orthologous tRNA/AATS pair
Example - p-amino Phe

Question 10

Homologs

Answer 10

Descended from common ancestor, similar sequence

Question 11

Paralogs

Answer 11

Proteins related via a gene duplication event, present in the same organism but different function

Question 12

Orthologs

Answer 12

Same gene and the same function, but different organsisms.

Question 13

Conformation of most peptide bonds

Answer 13

Trans

Question 14

Broadest + most restricted Ramachandran plot

Answer 14

Boadest = Gly, most restricted = pro

Question 15

Alpha helix capping

Answer 15

Capped at N terminus via H bond

Capped at C terminus due to Gly having an LH conformation

Question 16

Alpha helix forming ability

Answer 16

Dependent on delta Gu
Ala = most likely to form alpha helix
Pro + gly = least likely

Question 17

Why is Proline often found in 1st turn

Answer 17

Results in a 20 degree bend

Question 18

Where are amphipathic helices found?

Answer 18

Protein surface

Question 19

Polyproline helices

Answer 19

LH, no internal H bonds

Question 20

Antiparallel beta-sheets

Answer 20

One side is exposed the other buried, straight H bonds,, >2 strands

Question 21

Parallel beta-sheets

Answer 21

H bond at angle - less stable, >5 strands

Question 22

Globular Proteins

Answer 22

Compact structure, diverse range, hydrophobic core

Question 23

Motif

Answer 23

Ordered arrangement of secondary structure e.g. Helix-turn-helix (DNA binding protein)

Question 24

Why does leucine to alanine mutation in globular proteins have a large impact?

Answer 24

Leaves a large cavity - non-optimal core packing

Question 25

Types of protein cavity mutants

Answer 25

1. Steric mutants - shape of cavity changes, not volume 2. Extreme volume mutants - increase in volume of cavity 3. Polar mutants - polar residue in cavity

Question 26

Is protein surface normally polar or nonpolar?

Answer 26

Polar

Question 27

Effect of mutation creating many hydrophobic residues on protein surface?

Answer 27

Reduced solubility, protein aggregation

Question 28

Benefits of domains

Answer 28

Many protein folding faster, more efficent and can add function

Question 29

Subunit

Answer 29

1 polypeptide chain stably folded by itself

Question 30

Non-proteinogenic amino acids

Answer 30

GABA - neurotransmitter Histamine - immunological response Thyroxine - hormone (metabolism regulation)

Question 31

Non-standard aa found in proteins

Answer 31

Hydroxyproline | Lysine methylated by S-adenosylmethionine to create a less reactive version of the aa

Question 32

Enzyme on/off switch

Answer 32

Ser/Thr phosphorylation via O-glycosylation

Question 33

Non-specific protein modification

Answer 33

Random hydrolysis of arginine side chain can produce citrulline - cause of autoimmune disease.

Question 34

Measure of protein ageing

Answer 34

Glycation of protein amino groups

Question 35

In-vitro chemical modification

Answer 35

via site-directed mutagenesis e.g. amino group acetylation

Question 36

In vivo fluorescence labelling

Answer 36

Fusion via GFP gene

Question 37

In vitro fluorescence labelling

Answer 37

Use labels at different wavelengths

Question 38

Radioactive labelling evaluation [14C, 3H, 35S]

Answer 38

+ only require a few atoms, doesn't affect properties much | - Safety concerns

Question 39

Crosslinking reagents

Answer 39

Homobifunctional - both ends of the molecules react with the same functional group Heterobifunctional - different reactive groups at each end of the molecule - allows for more control

Question 40

Literature database

Answer 40

Pubmed

Question 41

Compound database

Answer 41

Pubmed compounds

Question 42

Sequence database

Answer 42

Genbank

Question 43

Functional protein information databank

Answer 43

UniprotKB

Question 44

3D Structure databank

Answer 44

CSD (small molecules), PDB (proteins)

Question 45

Protein Classes

Answer 45

1. Fold - same topology, no evidence for relation 2. Superfamily - same fold, related function, probably related 3. Family - structure and function very similar, almost defo related

Question 46

What does sequence alignment show?

Answer 46

Indels, gene duplication events, sequence similarity, synonymous and non-synonymous mutations

Question 47

Why is it easier to determine homology from protein sequence?

Answer 47

Each letter gives more information, protein evolves slower e.g. may be synonymous mutation - not shown in DNA sequence as clearly

Question 48

What % sequence required for homology?

Answer 48

>30%

Question 49

Types of alignment

Answer 49

1. Global alignment - search through an entire sequence looking for similarities 2. Local alignment - only searches through single domains 3. Pairwise alignment - only compares 2 sequences 4. Multiple alignment - more than 2 sequences

Question 50

Multiple sequence alignment

Answer 50

Used to generate phylogenetic trees Used to find characteristic protein family pattern Generated through tree method/divide and conquer method

Question 51

Types of phylogenetic tree

Answer 51

1. Cladogram - shows geneology, branch lengths arbitory 2. Additive tree - branch = measure of time 3. Ultrametric tree - always rooted and contains timeline

Question 52

E-value

Answer 52

no. expected hits by chance. Lower number = more relevant result

Question 53

Sliding window

Answer 53

Approach used to analyse amino acid properties - properties within a window are averaged, and then the window moves along sequence to build up full picture

Question 54

Protein function assigned based on...

Answer 54

1. Genome comparison 2. Sequence similarity - more likely in orthologs than paralogs 3. Structural similarity - specific fold linked to function