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Proteins Structure and Function > Lectures 4 - 7 > Flashcards

Lectures 4 - 7 Flashcards

1
Q

Ways to modify protein structure

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

Use of chemical reagents to modify protein structure

A

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

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3
Q

Use of unnatural amino acids to modify protein structure

A
  1. Allows a specific function to be added

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

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4
Q

SupE mutation in E.coli

A

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

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5
Q

Requirements to incorporate unnatural aa in vivo

A

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

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6
Q

Problems with incorporating unnatural aa in vivo

A

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

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7
Q

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

A
  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
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8
Q

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

A
  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
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9
Q

In vivo synthesis of unnatural aa

A

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

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10
Q

Homologs

A

Descended from common ancestor, similar sequence

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11
Q

Paralogs

A

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

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12
Q

Orthologs

A

Same gene and the same function, but different organsisms.

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13
Q

Conformation of most peptide bonds

A

Trans

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14
Q

Broadest + most restricted Ramachandran plot

A

Boadest = Gly, most restricted = pro

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15
Q

Alpha helix capping

A

Capped at N terminus via H bond

Capped at C terminus due to Gly having an LH conformation

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16
Q

Alpha helix forming ability

A

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

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17
Q

Why is Proline often found in 1st turn

A

Results in a 20 degree bend

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18
Q

Where are amphipathic helices found?

A

Protein surface

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19
Q

Polyproline helices

A

LH, no internal H bonds

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20
Q

Antiparallel beta-sheets

A

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

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21
Q

Parallel beta-sheets

A

H bond at angle - less stable, >5 strands

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22
Q

Globular Proteins

A

Compact structure, diverse range, hydrophobic core

23
Q

Motif

A

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

24
Q

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

A

Leaves a large cavity - non-optimal core packing

25
Q

Types of protein cavity mutants

A
  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
26
Q

Is protein surface normally polar or nonpolar?

A

Polar

27
Q

Effect of mutation creating many hydrophobic residues on protein surface?

A

Reduced solubility, protein aggregation

28
Q

Benefits of domains

A

Many protein folding faster, more efficent and can add function

29
Q

Subunit

A

1 polypeptide chain stably folded by itself

30
Q

Non-proteinogenic amino acids

A

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

31
Q

Non-standard aa found in proteins

A

Hydroxyproline

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

32
Q

Enzyme on/off switch

A

Ser/Thr phosphorylation via O-glycosylation

33
Q

Non-specific protein modification

A

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

34
Q

Measure of protein ageing

A

Glycation of protein amino groups

35
Q

In-vitro chemical modification

A

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

36
Q

In vivo fluorescence labelling

A

Fusion via GFP gene

37
Q

In vitro fluorescence labelling

A

Use labels at different wavelengths

38
Q

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

A

+ only require a few atoms, doesn’t affect properties much

- Safety concerns

39
Q

Crosslinking reagents

A

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

40
Q

Literature database

A

Pubmed

41
Q

Compound database

A

Pubmed compounds

42
Q

Sequence database

A

Genbank

43
Q

Functional protein information databank

A

UniprotKB

44
Q

3D Structure databank

A

CSD (small molecules), PDB (proteins)

45
Q

Protein Classes

A
  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
46
Q

What does sequence alignment show?

A

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

47
Q

Why is it easier to determine homology from protein sequence?

A

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

48
Q

What % sequence required for homology?

A

> 30%

49
Q

Types of alignment

A
  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
50
Q

Multiple sequence alignment

A

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

51
Q

Types of phylogenetic tree

A
  1. Cladogram - shows geneology, branch lengths arbitory
  2. Additive tree - branch = measure of time
  3. Ultrametric tree - always rooted and contains timeline
52
Q

E-value

A

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

53
Q

Sliding window

A

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

54
Q

Protein function assigned based on…

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

Proteins Structure and Function (5 decks)

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