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Proteins Structure and Function > Lectures 1 - 3 > Flashcards

Lectures 1 - 3 Flashcards

1
Q

Only aa to buffer at neutral pH

A

Histidine

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2
Q

Amino acid not in L form

A

Glycine - not chiral

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3
Q

Only modified aa incorporated during translation

A

SeCys

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4
Q

Which bonds can rotate in a peptide bond?

A

N-Calpha and Calpha-C

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5
Q

How is protein variety increased?

A

Gene duplication, Mosaic genes, De novo synthesis of non-coding DNA, horizontal gene transfer between species

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6
Q

Mosaic genes

A

Combine different parts of genes

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7
Q

The fate of duplicated genes

A

Loss, share function with old gene (subfunctionalisation), acquire a new function (neofunctionalization)

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8
Q

Role of protecting groups

A

Prevent unwanted side reactions. Fmoc protects the alpha-amino group, tboc protects side chain amino groups

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9
Q

Disadvantages with solution phase peptide synthesis

A

Low yield and time consuming

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10
Q

Kaiser test result

A

Used to test for free primary amine groups. A purple colour means that there are free primary amine groups = incomplete coupling. Yellow colour means that coupling is complete as there are no free amine groups.

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11
Q

Housekeeping Proteins

A

Required in all cells, all the time - they are essential for survival e.g. RNA pols, cytoskeletal proteins. Have long half life

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12
Q

Luxury Proteins

A

Tissue-specific, required for a specific function and only expressed when required. Have short half life.

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13
Q

Control of protein concentration at mRNA level

A

mRNA synthesis, PRM of mRNA, mRNA degradation

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14
Q

Control of protein concentration at protein level

A

protein synthesis, PTM of protein, protein targeting and transport, protein degredation

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15
Q

Control of protein concentration at cellular level

A

cell division

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16
Q

Coupling of transcription to translation

A

In eukaryotes but not in prokaryotes - delay of 1h

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17
Q

Why is genetic code more resistant to mutation?

A

Due to the degeneracy of the genetic code

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18
Q

Wobble base

A

3rd base in codon - has more steric freedom, meaning it can make an unconventional pairing . This gives resistance to mutations

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19
Q

Prokaryotic transcription steps

A

Only 1 RNA pol is present in prokaryotes, and genes have no introns. 10 nt upstream of promoter is an AT rich region called the Priebnow box. Sigma factor of Rpol binds to promoter and unwinds DNA helix in order to form a transcription bubble. Complementary RNA bases are added in the 5’ to 3’ direction. Transcription either stops due to a stop sequence being encountered or due to rho dependent/independent termination. Sigma factor is released, transcript released and DNA helix rewinds.

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20
Q

Eukaryotic transcription

A

Genes have introns. 3 RNA pols are involved:
RNA pol I = rRNA
RNA pol II = mRNA
RNA pol III = tRNA
AT region upsteam of promoter at -25nt called TATA box. Transcription factors bind to promoter and then RNA pol II can bind in order to form transcription initiation complex

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21
Q

Ribosome sizes

A

Prokaryotes = 70s, eukaryotes = 80s

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22
Q

Start codons

A 
Prokaryotes = N-formyl-methionine
Eukaryotes = methionine
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23
Q

Translation steps

A
  1. Amino acid activation
  2. Initiation
  3. Elongation
  4. Termination and ribosome recycling
  5. Folding and PTMs
24
Q

mRNA primary transcript processing

A

Addition of 5’ cap and 3’ poly A tail. Alternate splicing either via spliceosome or self-splicing

25
Q

Why can tRNA include unusual bases?

A

Due to enzymatic modifications

26
Q

Differences between aminoacyl-tRNA synthetases - class I and II

A

Different oligomerisation states
Different conformation of bound ATP
Different tRNA binding face
Different site of amino acid attachment

27
Q

Class I synthetases

A

Transfer aminoacyl group on to 2’OH on 3’ terminal A residue to tRNA and AMP released. Transesterification moves aminoacyl group to 3’ OH in order to form aminoacyl tRNA

28
Q

Clas II synthetases

A

Aminoacyl group directly tranfered on to the 3’ OH and AMP released

29
Q

Translation Initiation

A

IF-1 and IF-3 bind to 30S ribosomal subunit. This binds to mRNA and scans for the start codon. When start codon is reached, initiator tRNA bound to IF-2 and GTP binds to start codon. This allows large subunit to associate with small subunit to form full ribosome. All IFs dissociate and GTP hydrolysed to GDP and Pi

30
Q

Reasons for protein degredation

A

Prevents accumulation of misfolded proteins, allows for protein regulation and adaptation to changing environments, allows amino acid recycling.

31
Q

Types of protein degredation

A

Selective ATP-dependent cytosolic system and non-selective lysosomal system.

32
Q

Task: Draw ubiquitin tagging pathway

A

Check notes

33
Q

26S proteosome components

A

Lid is responsible for substrate recognition and deubiquitination. Base for unfolding, substrate translocation and has AAA+ protease.

34
Q

AAA+ protease

A

Hexameric ring with a very small pore. Contains regulatory subunits for sustrate recognition. Binding of ATP results in a conformational change which allows the protein to unfold, resulting in the protein entering the pore for degredation

35
Q

Different E3 classifications

A

HECT
RING
RBR

36
Q

Degrons

A

Degredation signals involved in recognition. Work via:
Binding to AAA and hexamer pore
Binding to auxillary sites on pore
Mediating reactions that allow protease recognition

37
Q

N-end rule pathway

A

t1/2 depends on N-terminal aa exposed after co-translational cleavage of N-terminal Met. N-recognin binds and ubiquitinates protein if the aa is a primary destabilising residue. Secondary/tertiary destabilising residues are modified via enzymes to form primary residue

38
Q

PEST sequnce

A

High in P, E, S, T. Have short t1/2 and degraded via unknown mechanism.

39
Q

Ways to separate molecules

A
  1. Different molecular migration velocity under specific field e.g. electrophoresis/sedimentation
  2. Different partitioning between different phases e.g. phase separation
40
Q

Separation Parameters

A

Size e.g. gel chromatography
Charge e.g. ion exchange chromatography
Hydrophobicity e.g. precipitation

41
Q

Native Protein Precipitation Methods

A

Salt e.g. Ammonium chloride - cheap, gentle but may require desalting
Organic solvent - cheap, but may denature protein
Organic polymer, expensive but will not denature protein

42
Q

Batch Adsorption

A

Adsorbent added to solution, desired molecule adsorbs to material. Supernatant removed. Component removed from adsorbent via a new buffer.

43
Q

Gel Chromatography

A

Separates based on molecule size. A column is packed with porous gel beads. The large molecules run straight through the gel, but the small molecules get stuck in the gel pores and take longer to run through.

44
Q

Ion exchange chromatography

A

Separates based on molecular charge. Gel is substituted with charged groups - for anion exchange DEAE and for cation exchange CM groups. The charged group in the molecule will bind to the oppositely charged molecule in the gel. The uncharged molecules will flow straight through the gel.

45
Q

Elution strategies

A

Isocratic elution - fixed buffer conditions
Gradient elution - constantly change the buffer conditions
Ionic strength increase - creates competition for binding
pH change - changes the interactions

46
Q

Hydrophobic interaction chromatography

A

Hydrophobic groups are substituted onto a gel. Hydrophobic regions on proteins will bind to these groups. Polar organic solvents can then be used to weaken the hydrophobic effect.

47
Q

Affinity chromatography

A

Based on specific protein properties. Bioaffinity based on biological interactions - can use enzyme inhibitor, specific antibodies. Would use glutathione for GST/ AMP for nucleotide using enzymes.

48
Q

Salt precipitation evaluation

A

+ Works at any scale and produces a concentration 1st product
- Requires desalting before ion exchange chromatography

49
Q

Ion Exchange Chromatography Evaluation

A

+ Low cost, efficient, can be reused at different pH to change parameter
- Can only separate based on one change at a time

50
Q

Gel Chromatography Evaluation

A

+ Don’t need to change buffer after salt precipitation and homologous proteins grouped together
- Low capacity

51
Q

Hydrophobic Interactions Chromatography Evaluation

A

+ Molecules can be + or - and has a good capacity

- Not all molecules adsorb

52
Q

Affinity Chromatography Evaluation

A

+ very specific, therefore can get minor components in a good concentration
-costly and not always applicable

53
Q

Affinity Tag

A

Must not interfere with function or can be easily removed e.g. His tag removed with imidazol

54
Q

To assess purity

A

General composition via analytic electrophoresis of high performance chromatography. To quantify a specific component use an immunological/activity assay - doesn’t show impurities.

55
Q

Types of charactersation

A

Physical - mass, size, charge properties
Chemical e.g. mass spectrometry, amino acid composition
Biological characteristaion e.g. enzyme activity/in vivo effects

Proteins Structure and Function (5 decks)

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