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BMS2062 > PART IV: PROTEIN EXPRESSION AND MODIFICATION > Flashcards

PART IV: PROTEIN EXPRESSION AND MODIFICATION Flashcards

1
Q

What is the protein folding problem known?

A
  • How does a protein’s aa sequence manage to fold into a 3D structure?
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2
Q

Why did Anfinsen choose to study the Bovine Panreatic Ribonuclease A for the protein folding problem? (3 reasons)

A
  • Because it is small (124 aa)
  • Contains 8 Cys residues
  • Can degrade ribonucleic acid (enzymatic activity) –> use to measure the % activity
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3
Q

What does the substance BME do?

A
  • Breaks the disulfide bonds
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4
Q

what does urea do?

A
  • Denatures proteins by binding with H bonds thus inhibiting the bonds
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5
Q

In the Anfinsen experiments, what happened when BME was removed and urea was present?

A
  • Only 1% of activity was reformed from the denatured protein
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6
Q

What does the result of only 1% of the denatured protein refolding after BME (disulfide bond breaker) was removed mean?

A
  • That you need the rest of the protein to be in the correct conformation to be functional
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7
Q

In the Anfinsen experiments, what happened when both the urea and BME were removed?

A
  • There was 100% folding and functionality again
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8
Q

Why does the native structure of proteins form naturally?

A
  • Due to the Gibbs free energy (lowest energy state)
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9
Q

What is the thermodynamic hypothesis (Anfinsen)?

A
  • that the 3D structure of a protein is what gives it the lowest delta G
  • There are multiple intermediate stages to protein folding
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10
Q

Is the thermodynamic hypothesis valid for proteins without disulfide bonds?

A
  • YES
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11
Q

Is the structure of a protein more conserved than sequence?

A
  • YES

- Native state can be stabilised by a small number of conserved interactions

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12
Q

Approximately how many amino acids are conserved across evolution?

A
  • Approx 5 aa (2 Tryp and 1 Pro)
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13
Q

Are lots or only a few amino acids crucial for a proteins structure?

A
  • Only a few e.g. Beta strand structure
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14
Q

Can sequences of low similarity have the same 3D structure?

A
  • YES!

- Different amino acids can code for the SAME strucutral domain

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15
Q

What are the two forms of predictions if conserved interactions are broken?

A
  • Independent interactions (Stepwise ladder on graph)

- Cooperative (sigmoidal shape on graph)

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16
Q

What are 4 situations in which the Thermodynamic hypothesis may not be valid?

A
  • Some proteins aggregate under certain conditions
  • In vivo folding may require accessory proteins (chaperones)
  • Zymogens –> Fold then are cleaved, mature protein may be ‘trapped’ in zymogen-like fold e.g. alpha-lytic protease
  • Some proteins have more than one “native” structure
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17
Q

What are three reasons to consider the protein folding?

A
  1. Get protein structure prediction from protein databases
  2. Protein design/engineering and drug design
  3. Understand the molecular basis of diseases
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18
Q

What is thought to be the type of misfolding in Alzheimers, Huntingtons and Parkinsons?

A
  • Amyloidosis–> formation of amyloid fibrils with highly ordered Beta strands
  • Amyloid protein aggregates in weird ways and induces the misfolding of other proteins `
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19
Q

What is a proteome defined as?

A
  • A set of proteins produced in an organism, system, or biological context
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20
Q

What is proteomics?

A
  • Large scale study of proteomes
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21
Q

Does the quantity of RNA level and subsequent protein level matter?

A
  • YES it does
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22
Q

What are 3 factors that influence protein levels?

A
  1. Localisation
  2. Damage and/or Degradation
  3. Post translational Modifications (PTM)
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23
Q

What does localisation of proteins involve (strucutres -4 of them)?

A
  • Subcellular organelles
  • Multiprotein complexes
  • Membranes
  • Extracellular spaces
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24
Q

What does damage and/or degradation involve in proteins?

A
  • Heat shock
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25
Q

What does PTMs in proteins invovle? (8 PTMS)

A
  • Proteolytic cleavage
  • Acetylation
  • Merthylation
  • Phosphorylation
  • Ubiquitination
  • Sulfation
  • Selenoproteins
  • Gylcosylation
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26
Q

What does Phosphorylation usually change in a protein?

A
  • The shape
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27
Q

What type of PTM (which molecule) do Plasma membrane proteins carry?

A
  • N-glycans
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28
Q

What type of PTM (which molecule) do Nuclear and cytoplasmic proteins carry?

A
  • O-glycans
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29
Q

What occurs in the Phosphorylation PTM?

A
  • Introduces a -ve charged and hydrophillic group
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30
Q

What can cancer be caused by in proteins?

A
  • By the alterations in the PTMs of proteins

e. g. Histones, NFkB, signalling proteins)

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31
Q

What are seven things that proteomics is good for?

A
  1. Obtaining the most complete possible information about the components of cells
  2. To determine the presence and effects of protein modifications
  3. Systems biology–> holistic view of the network of interactions leading to biological phenotypes
  4. Identification of cell-type markers
  5. Identification of disease markers
  6. Identification of potential drug targets
  7. tracking the progress of disease or therapy
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32
Q

What does personalised medicine allow for?

A
  • The biomarkers to be targeted that someone has

- Narrow down the best treatment to target individual diagnosis (proteome of human cancer)

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33
Q

Do some anti cancer drugs accidentally target the normal instead of the diseased protein?

A
  • YES

- This can lead to nasty side effects

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34
Q

What are the three different types of proteomics?

A
  • Expressional prtoeomics
  • Functional proteomics (limited protein sets to determine function–> interacting proteins in vivo)
  • Structural proteomics ( high-throughput determination of protein structure)
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35
Q

What does expressional proteomics involve and what 3 questions are asked?

A
  • Studying complete proteomes (from two deferentially treated cell lines)
  • e.g. What proteins are present?
    What are their expression levels?
    What are their PTMs?
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36
Q

What are the steps in mass spec proteomics?

A
  • EXTRACT proteins from biological sample
  • TRYPSIN DIGESTION into peptides
  • SEPARATE proteins from each other to ENRICH proteins with specific PTM
  • Determine the IDENTITIES of the proteins (obtain partial sequences by Mass spec, identify full sequences from databases)
  • Determine QUANTITIES of proteins
  • Verify PTMS (separate proteins out based on PTM type)
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37
Q

Which kind of PTM occurs on Tyrosine residues and ads negative charges to the protein?

A
  • Phosphorylation (but can also be on Serine or threonine)
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38
Q

What are the cleavage points for trypsin?

A
  • Lys and Arg
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39
Q

What are the cleavage points for chymotrypsin?

A
  • Phe, Trp and Tyr
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40
Q

What are the cleavage points for Pepsin?

A
  • Leu, Phe, Tryp, Tyr
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41
Q

What is the first dimension in 2D gel electrophoresis?

A
  • Separated according to molecular weights (the second step in procedure)
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42
Q

What is the second dimension in 2D gel electrophoresis?

A
  • The spots migrate according to the pI (voltage) (first step in the process)
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43
Q

What are the two steps to 2D gel electrophoresis?

A
  1. Separating according to pI (charge)

2. Separation acccording to molecular weight

44
Q

In 2D gel electrphoresis, a protein will move to the position according to its ___?

A
  • pI (charge)

- Travel until they reach the neutral charge (when pI= environmental pH)

45
Q

In 2D gel electrophoresis, is the low pH at the positive or negative charge?

A
  • Low pH at the positive charge
46
Q

Does a high pI have a positive or negative charge in 2D gel electrophoresis?

A
  • Has a negative charge

- (and the low pI has a positive charge)

47
Q

What are two different ways of using mass spectrometry to identify “spots”?

A
  1. Determine the mass of a protein (or digest and determine the masses of peptides–> compare to predicted masses from gene sequences
  2. Sequence the peptides in mass spectrometer (compare to predicted sequences from gene/EST sequences)
48
Q

What types of PTMs do chemokine receptors have?

A
  • Disulfide bonds
  • Tyrosine sulfation sites
  • N-linked glycosylation sites
49
Q

Where does tyrosine sulfation occur?

A
  • In the trans golgi network
50
Q

Why does tyrosine sulfation only occur in secreted or cell surface proteins?

A
  • Because enzymes that add the sulfate group exist in the trans-golgi network
51
Q

Is tyrosine sulfation known to be dynamically regulated (reversible)?

A
  • It is not know (not sure)
52
Q

What does tyrosine sulfation regulate?

A
  • Protein-protein interactions
53
Q

What are tyrosylprotein Sulfotransferases? (TPSTs)

A
  • Coenzymes that catalyse the transfer of sulfate from PAPS (like ATP) to Tyr
54
Q

Which environment are TPSTs selective for Tyrosine in?

A
  • Acidic environment

- i.e. neighboring aas are acidic e.g. Aspartic acid and glutamic acid

55
Q

What are the two human isoforms of TPST?

A
  • TPST-1 and 2
56
Q

What does the knockout of mice orthologues (TPSTs) cause?

A
  • reduced body weight (feeding digestion problems)
  • Reduced fertility (sperm motility)
  • Early death (in a double knockout)
57
Q

What can the mutation of sulfation sites cause in terms of the chemokine response?

A
  • A reduced chemokine response
58
Q

Which mutation do individuals who are resistant to HIV have?

A
  • Mutation in CCR5 (chemokine receptor 5)
59
Q

If an experiment is done to show whether CCR5 is sulfated or not and cells were grown in the presence of cysteine or methionine –> then the protein had either cysteine, methionine, or both, is this an example of a positive or negative control?

A
  • Positive control
60
Q

What does immunoprecipitation involve (context of CCR5 experiment)?

A
  • Treat a sample that contains a sample of proteins with an Ig that is targeted to ONE of the proteins–> Ig must be recognised by beads that can PULL it out of solution.
  • Performed an immunoprecipitation targeted to CCR5
61
Q

What is DARC and what does it stand for?

A
  • Duffy Antigen Receptor for Chemokine
  • “decoy” chemokine receptor
  • Defines the blood type (A/B/O)
62
Q

Where is DARC expressed?

A
  • On erythrocytes
63
Q

Is DARC G protein coupled?

A
  • NO
64
Q

What does chemokine binding cause for the chemokine receptor?

A
  • Internalisation

- Removal of the chemokine from the circulation

65
Q

What is an example of the DARC in use?

A
  • Malarial parasite (Plasmodium vivax) uses DARC to infect RBCs
66
Q

Are both tyrosine (Y) residues in the N terminus of DARC both sulfated?

A
  • YES
67
Q

What is the basis for the drug Maraviroc in HIV?

A
  • that individuals that were resistant to certain forms of HIV had higher levels of chemokines (in serum and certain chemokines)
  • these chemokines can bind to and block the binding by HIV –> drug development
68
Q

What are enzymes often named based on?

A
  • The reaction they cataylse

- Combines the substrate and ‘ase’ e.g. amylase (digests starch-amylin)

69
Q

What environment does the enzymes active site provide?

A
  • One that is energetically more favourable
70
Q

If 636 kcal/mole of energy is released (enough to set off an explosion) through the oxidation of glucose to CO2, then why doesn’t glucose spontaneously combust?

A
  • Because there is an activation energy that it must overcome (in form of heat)
71
Q

What are 6 characteristics of enzymes?

A
  1. They work RAPIDLY
  2. Activity is dependent on pH (bc. aa like to be protonated)
  3. Activity dependent on temp
  4. Enzymes are SUBSTRATE SPECIFIC
  5. Many enzymes require cofactors or coenzymes (e.g. Fe, Zn and NAD+ respectively)
  6. Enzymes can be INHIBITED
72
Q

What is an example of enzymes bringing reactants close together?

A
  • Spliceosome
73
Q

How to enzymes lower the activation energy of a reaction?

A
  • They stabilise the transition state of a reaction
74
Q

What is the transition state?

A
  • The moment when the bonds are broken and reformed (corresponds to the top of the energy barrier)
75
Q

Does the transition state have a lifetime?

A
  • NO because it is not a stable chemical species

- (lifetime is infinitesimally small)

76
Q

Does the transition state make better contacts with the enzyme compared with the substrate or product does?

A
  • YES
77
Q

What concept is key for drug design in terms of enzymes?

A
  • Mimicing the reaction transition state (hence strong bonding to the enzyme)
78
Q

Is a nucleophile an electron rich or poor atom?

A
  • Electron rich (it wants to donate)
79
Q

Is an electrophile an electron rich or poor atom?

A
  • Electron poor (it wants to accept)
80
Q

What is a nucleophillic attack known as?

A
  • When a nucelophile will be attacking an electron defficient atom (electrophile)
81
Q

What are the two general steps in the chymotrypsin cleavage of the peptide?

A
  1. Formation of the acyl-enzyme intermediate

2. Hydrolysis of acyl enzyme intermediate

82
Q

What is the reason why we have such a detailed understanding of the mode of action of the enzyme structure saffold?

A
  • Due to the availability of crystal structures with substrates, products and intermediates
83
Q

Enzyme structure is a scaffold that _______ and ______?

A
  • Holds the catalytic functional groups in position

- Creates a substrate binding cleft that dictates specificity for a particular substrate

84
Q

What have mutations in the chymotrypsin gene shown?

A
  • Shown pancreatic damage in patients from high levels of active trypsin
85
Q

What does chymotrypsin play an important role in ?

A
  • The degradation of trypsinogen in the pancreas (digestive enzyme but plays role in regulation of other digestive enzymes)
86
Q

Are there a finite number of protein folds?

A
  • Yes
87
Q

Are protein domains units of compact structure, evolved function, and autonomous fold?

A
  • YES
88
Q

Will each domain of a protein match to a search in the database where there will be a function for an independent protein?

A
  • YES
89
Q

What is the definition of a protein domain?

A
  • Spatially distinct structural component that could conceivably fold and function in ISOLATION
90
Q

Can one protein be comprised of several domains?

A
  • YES
91
Q

Will each domain in a protein fold independently before the next?

A
  • YES
92
Q

In a protein model depiction what does the blue colour generally mean?

A
  • +ve charge
93
Q

In a protein model depiction what does the red colour generally mean?

A

-ve charge

94
Q

What has the SH2 domain evolved to recognise?

A
  • phosphotyrosines, approx 100 residues long
95
Q

All SH2 domains will bind pTyr but differ with respect to _______?

A
  • Their target recognition
96
Q

What is the RNA recognition motif (RRM)?

A
  • Binds to RNA across face of a beta-sheet (at least 4 beta strands)
  • Different RRMs have specificity for different RNA sequences
97
Q

Why does the RRM have strategically placed aromatic residues?

A
  • to interact with bases on RNA (these are aromatic residues)
98
Q

Which residues do you need to look for if a protein is a RNA binding protein (RBP)?

A
  • Tyrosine (Y) or Phenylalanine (F)
99
Q

Why does the PolyA tail allow for the post-mRNA to be protected from degradation?

A
  • Because the Poly A binding protein binds to it (PABP)
100
Q

What are the details of PABP (Poly A Binding Protein)?

A
  • 71 kDa RNA binding protein
  • 4 RNA recognition motifs (RRM) and C terminal domain (MLLE)
  • Has specificity for poly-A -tracts
101
Q

What two main things can we learn about domains from multiple sequence alignments?

A
  • Conserved residues can be identified

- Amino acid sequence identity of approx. 30% strongly suggests proteins have the same fold

102
Q

What is the threshold for sequence alignments that indicates that proteins contain the same fold?

A
  • 30% identity OR MORE (25% at a stretch )
103
Q

If two proteins look strucutrally similar, does that mean that they will have a high sequence similarity?

A
  • NO

- e.g. convergent evolution results in proteins with similar folds but no significant sequence identity

104
Q

What are zinc fingers?

A
  • small protein strucutral motif (domain) that is characterized by the coordination of one or more zinc ions to stabilise the fold. (molecular glue that holds domain together)
105
Q

Where are zinc finger domains found?

A

-TFs e.g. Transcripton factor IIIA

106
Q

Where do the zinc finger domains usually insert?

A
  • Into the main groove of DNA

BMS2062 (7 decks)

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