Protein Expression & Modification

Question 1

Which of the following is the correct order of the molecular masses of the three proteins A, B and C?

Select one:

A < B < C

A < C < B

B < A < C

C < A < B

C < B < A

Answer 1

Which of the following is the correct order of the molecular masses of the three proteins A, B and C?

Select one:

A < B < C

A < C < B

B < A < C

C < A < B

C < B < A

Question 2

The presence of an intense band at ~38 kDa in lane 2 but not in lane 1 indicates that…

Select one:

The cells express CD4 even without being transfected with the expression vector

The cells express CCR5 even without being transfected with the expression vector

The cells express CD4 only after transfection with the expression vector

The cells express CCR5 only after transfection with the expression vector

The cells express both CD4 and CCR5 only after transfection with the expression vector

Answer 2

The presence of an intense band at ~38 kDa in lane 2 but not in lane 1 indicates that…

Select one:

The cells express CD4 even without being transfected with the expression vector

The cells express CCR5 even without being transfected with the expression vector

The cells express CD4 only after transfection with the expression vector

The cells express CCR5 only after transfection with the expression vector

The cells express both CD4 and CCR5 only after transfection with the expression vector

Question 3

Definition

positively charged or neutral species having vacant orbitals that are attracted to an electron rich centre

Answer 3

Electrophile

Question 4

Sulfation ________ binding to chemokines and to pathogen proteins

Answer 4

Sulfation enhances binding to chemokines and to pathogen proteins

Question 5

Define

Poly-A-binding protein

Answer 5

a RNA-binding protein which binds to the poly(A) tail of mRNA

Question 6

What are the three types of proteomics?

Answer 6

Expressional proteomics

Functional proteomics

Structural proteomics

Question 7

Define

Wobble position

Answer 7

the 3rd nucleotide in a codon. Binding of a codon in an mRNA the cognate tRNA is much “looser” in the third position of the codon

Question 8

Definition

a putative RNA-binding domain of about 90 amino acids that are known to bind single-stranded RNAs

Answer 8

RNA-recognition motif

Question 9

Why is proteomics important?

Answer 9

1. To obtain the most complete possible information about the components of cells
2. To determine the presence and effects of protein modifications
3. Systems biology - a “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

Question 10

Why do we need to identify phosphorylated proteins in the cell?

Answer 10

Phosphorylation cascades are involved in many signalling pathways

Question 11

What two things can mass spec identify in proteins?

Answer 11

1. Determine mass of protein
2. Sequence peptides

Question 12

Which type of PTM introduces a negatively charge and hyrophilic group?

Answer 12

Phosphorylation

Question 13

Which of the following is the correct order of the charges of the three proteins A, B and C (going from least to most positively charged at the same pH value)?

Select one:

A < B < C

A < C < B

B < A < C

C < A < B

C < B < A

Answer 13

Which of the following is the correct order of the charges of the three proteins A, B and C (going from least to most positively charged at the same pH value)?

Select one:

A < B < C

A < C < B

B < A < C

C < A < B

C < B < A

Question 14

True or False:

The same regio that bonds chemokines also binds malaria

Answer 14

True

Question 15

Which position is denoted the “wobble position”?

Answer 15

Third nucleotide in a codon

Question 16

What PTMs do chemokine receptors have?

Answer 16

Tyrosine sulfation

N-linked glycosylation

Phosphorylation

Question 17

In normal brain tissues, the lysine residue at amino acid position 315 of the transcription factor, NF-κB, is not methylated. However, in a rare type of brain tumour, lysine 315 of NF-κB is methylated. Note that apart from lysine 315, NF-κB is post-translationally modified by methylation, phosphorylation, acetylation and ubiquination at a large number of other amino acid residues. You have been given the brain tissue lysate of an individual to analyse using a proteomic approach.

Which of the following is the most effective way to determine whether the brain tissue lysate contains the post-translationally modified form of NF-κB that is methylated at lysine 315?

Select one:

Mass spectrometry → isoelectric focusing → 2D gel electrophoresis

Immunoprecipitation using an antibody that specifically recognizes methylated lysine → 2D gel electrophoresis → N-terminal sequencing by Edman degradation

Isoelectric focusing → 2D gel electrophoresis → BLAST search

Digestion with trypsin → immunoprecipitation using an antibody that specifically recognizes methylated lysine → tandem mass spectrometry (MS/MS)

Answer 17

In normal brain tissues, the lysine residue at amino acid position 315 of the transcription factor, NF-κB, is not methylated. However, in a rare type of brain tumour, lysine 315 of NF-κB is methylated. Note that apart from lysine 315, NF-κB is post-translationally modified by methylation, phosphorylation, acetylation and ubiquination at a large number of other amino acid residues. You have been given the brain tissue lysate of an individual to analyse using a proteomic approach.

Which of the following is the most effective way to determine whether the brain tissue lysate contains the post-translationally modified form of NF-κB that is methylated at lysine 315?

Select one:

Mass spectrometry → isoelectric focusing → 2D gel electrophoresis

Immunoprecipitation using an antibody that specifically recognizes methylated lysine → 2D gel electrophoresis → N-terminal sequencing by Edman degradation

Isoelectric focusing → 2D gel electrophoresis → BLAST search

Digestion with trypsin → immunoprecipitation using an antibody that specifically recognizes methylated lysine → tandem mass spectrometry (MS/MS)

Question 18

Definition

a very short-lived configuration of atoms at a local energy maximum in a reaction-energy diagram

Answer 18

Transition state

Question 19

Why does the enzyme are the product dissociate?

Answer 19

They have low affinity for one another

Question 20

True or False:

PTMs can both enhance or inhibit protein function

Answer 20

True

Question 22

Define

Nucleophile

Answer 22

a molecule or substance that has a tendency to donate electrons or react at electron-poor sites such as protons.

Question 23

What does Tyrosylprotein Sulfotransferases catalyse?

Answer 23

Catalyse transfer of sulfate from PAPS to tyr

Question 24

Definition

a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides

Answer 24

Chymotrypsin

Question 25

Which regions of chemokine receptors are sulfated?

Answer 25

N-terminus

Question 26

Inhibitors that mimic the reaction transition state show very strong/weak binding to the enzymes

Answer 26

Inhibitors that mimic the reaction transition state show very strong binding to the enzymes

Question 27

Definition

a non-protein compound that is necessary for the functioning of an enzyme

Answer 27

Coenzyme

Question 28

The oxidation of glucose to CO₂ and H₂O releases a massive amount of free energy in the form of heat. Why doesn’t glucose spontaneously combust?

Answer 28

Although powdered glucose is highly flammable, it requires input of activation energy in form of heat (i.e. the energy barrier is high)

Question 29

Why does the native structure of a protein form spontaneously?

Answer 29

The native structure is thermodynamically favoured. i.e. it has the lowest Gibbs free energy

Question 30

Define

Phosphorylation

Answer 30

the chemical addition of a phosphoryl group (PO3-) to an organic molecule

Question 31

The most variable region of the sequence alignment is indicated by “less than” signs (<). What is the likely role of this region?

Select one:

Forming hydrophobic interactions within the protein core

Forming salt bridges (ionic interactions) within the protein core

Forming salt bridges (ionic interactions) with residues in the corresponding receptors

Forming disulfide bonds

Forming one or more loop structure(s)

Answer 31

The most variable region of the sequence alignment is indicated by “less than” signs (<). What is the likely role of this region?

Select one:

Forming hydrophobic interactions within the protein core

Forming salt bridges (ionic interactions) within the protein core

Forming salt bridges (ionic interactions) with residues in the corresponding receptors

Forming disulfide bonds

Forming one or more loop structure(s)

Question 32

Definition

a molecular entity that is formed from the reactants (or preceding intermediates) and reacts further to give the directly observed products of a chemical reaction

Answer 32

Reaction intermediate

Question 33

What is Functional proteomics?

Answer 33

Studies more limited protein sets to determine their function

e.g. identify interacting proteins in vivo

Question 34

Definition

a branch of biotechnology concerned with applying the techniques of molecular biology, biochemistry, and genetics to analyzing the structure, function, and interactions of the proteins produced by the genes of a particular cell, tissue, or organism, with organizing the information in databases, and with applications of the data

Answer 34

Proteomics

Question 35

In normal brain tissues, the lysine residue at amino acid position 315 of the transcription factor, NF-κB, is not methylated. However, in a rare type of brain tumour, lysine 315 of NF-κB is methylated. Note that apart from lysine 315, NF-κB is post-translationally modified by methylation, phosphorylation, acetylation and ubiquination at a large number of other amino acid residues. You have been given the brain tissue lysate of an individual to analyse using a proteomic approach.

The results of your analysis show that the given brain tissue lysate contains NF-κB that is not methylated at lysine 315 but the concentration of NF-κB present is 100 times higher than that found in healthy individuals. Based on this observation, you conclude that…

Select one:

the individual has developed the rare type of brain tumour

the individual is highly unlikely to develop any cancer

the individual may not have developed the rare type of brain tumour but has a relatively high risk of developing cancers

the individual is healthy and has a relatively low risk of developing cancers

Answer 35

In normal brain tissues, the lysine residue at amino acid position 315 of the transcription factor, NF-κB, is not methylated. However, in a rare type of brain tumour, lysine 315 of NF-κB is methylated. Note that apart from lysine 315, NF-κB is post-translationally modified by methylation, phosphorylation, acetylation and ubiquination at a large number of other amino acid residues. You have been given the brain tissue lysate of an individual to analyse using a proteomic approach.

The results of your analysis show that the given brain tissue lysate contains NF-κB that is not methylated at lysine 315 but the concentration of NF-κB present is 100 times higher than that found in healthy individuals. Based on this observation, you conclude that…

Select one:

the individual has developed the rare type of brain tumour

the individual is highly unlikely to develop any cancer

the individual may not have developed the rare type of brain tumour but has a relatively high risk of developing cancers

the individual is healthy and has a relatively low risk of developing cancers

Question 36

Define

Catalytic residues

Answer 36

the amino acids directly involved in chemical catalysis. They mainly act as a general acid–base, electrophilic or nucleophilic catalyst or they polarize and stabilize the transition state

Question 37

Why can we not deduce anything about sulfation based on Lanes 1-4?

Answer 37

We cannot deduce anything about sulfation since they were labelled non-specifically (i.e. with ³⁵S-Cys/Met)

Question 38

What does the addition of Mercaptoethanol (BME) do to a protein?

Answer 38

Denatures it by breaking disulphide bonds

Question 39

Which features of proteins are used for 1D Gel electrophoresis?

Answer 39

Size

Question 40

Definition

a large protein family of receptors that detect molecules outside the cell and activate internal signal transduction pathways and, ultimately, cellular responses

Answer 40

G-protein-coupled receptors

Question 41

Is the Thermodynamic Hypothesis valid for proteins without disulphide bonds?

Answer 41

Yes

Question 42

What recognises stop codons?

Answer 42

Release factors

Question 43

The three-dimensional structure of a native protein in its normal physiological environment is the one in which the Gibbs free energy of the while system is what?

Answer 43

The lowest

Question 44

Which is the most suitable conclusion that can be drawn from the experiment?

Select one:

Transfected cells do not express CCR5 in the presence of sulfate

Transfected cells do not express CD4 in the presence of sulfate

Sulfate blocks the expression of CCR5 by non-transfected cells

Sulfate blocks the expression of CD4 by non-transfected cells

Non-transfected cells do not express either CCR5 or CD4

Answer 44

Which is the most suitable conclusion that can be drawn from the experiment?

Select one:

Transfected cells do not express CCR5 in the presence of sulfate

Transfected cells do not express CD4 in the presence of sulfate

Sulfate blocks the expression of CCR5 by non-transfected cells

Sulfate blocks the expression of CD4 by non-transfected cells

Non-transfected cells do not express either CCR5 or CD4

Question 45

The presence of intense bands at ~38 kDa in lane 2 AND at ~54 kDa in lane 4 but no corresponding bands in lanes 1 and 3 indicates that…

Select one:

The cells express CD4 even without being transfected with the expression vector

The cells express CCR5 even without being transfected with the expression vector

The cells express CD4 only after transfection with the expression vector

The cells express CCR5 only after transfection with the expression vector

The cells express both CD4 and CCR5 only after transfection with the expression vector

Answer 45

The presence of intense bands at ~38 kDa in lane 2 AND at ~54 kDa in lane 4 but no corresponding bands in lanes 1 and 3 indicates that…

Select one:

The cells express CD4 even without being transfected with the expression vector

The cells express CCR5 even without being transfected with the expression vector

The cells express CD4 only after transfection with the expression vector

The cells express CCR5 only after transfection with the expression vector

The cells express both CD4 and CCR5 only after transfection with the expression vector

Question 46

Mass spectrometry analysis demonstrated that the two proteins in box D have exactly the same amino acid sequence. Which of the following is the most likely explanation for them occurring at different positions in the gel, as shown?

Select one:

They have substantially different expression levels.

They are encoded by different mRNA splice variants.

One of them is post-translationally modified with a charged group.

One of them is post-translationally modified with a group that substantially increases its molecular weight.

One of them contains disulfide bonds whereas the other does not.

Answer 46

Mass spectrometry analysis demonstrated that the two proteins in box D have exactly the same amino acid sequence. Which of the following is the most likely explanation for them occurring at different positions in the gel, as shown?

Select one:

They have substantially different expression levels.

They are encoded by different mRNA splice variants.

One of them is post-translationally modified with a charged group.

One of them is post-translationally modified with a group that substantially increases its molecular weight.

One of them contains disulfide bonds whereas the other does not.

Question 47

Which of the following base pairs can form between the first position of a codon and the third position of the corresponding anticodon?

Select one:

A:G

A:U

A:C

C:I

U:C

Answer 47

Which of the following base pairs can form between the first position of a codon and the third position of the corresponding anticodon?

Select one:

A:G

A:U

A:C

C:I

U:C

Question 48

Chymotrypsin catalyses the cleavage of peptide bonds on which terminal of which amino acids?

Answer 48

Cleavage of peptide bonds on the carboxy-terminal side of Phe, Trp and Tyr residues

Question 49

In the classic experiment by Christian Anfinsen, Anfinsen began with an aqueous solution containing purified, fully active ribonuclease (RNaseA) in a simple buffer with no other additives. The sample was first treated with urea and beta-mercaptoethanol (BME), resulting in the complete loss of RNase A catalytic activity. Subsequently, the urea and BME were removed and the sample was found to regain 100% of its original catalytic activity.

What was the role of BME in the Anfinsen experiment?

Select one:

To catalyse the folding of RNase A

To decrease the free energy of the folded protein

To destabilise the protein structure

To form the disulfide bonds

To break the disulfide bonds

Answer 49

In the classic experiment by Christian Anfinsen, Anfinsen began with an aqueous solution containing purified, fully active ribonuclease (RNaseA) in a simple buffer with no other additives. The sample was first treated with urea and beta-mercaptoethanol (BME), resulting in the complete loss of RNase A catalytic activity. Subsequently, the urea and BME were removed and the sample was found to regain 100% of its original catalytic activity.

What was the role of BME in the Anfinsen experiment?

Select one:

To catalyse the folding of RNase A

To decrease the free energy of the folded protein

To destabilise the protein structure

To form the disulfide bonds

To break the disulfide bonds

Question 50

What are the steps to 2D Gel Electrophoresis?

Answer 50

1. Separation accoridng to pI (charge)
   
   • Proteins travel until neutrally charged
2. Separation according to molecular weight

Question 51

Define

Chymotrypsin

Answer 51

a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides

Question 52

Which part of the RNA-recognition motif binds to RNA?

Answer 52

The face of the beta sheet

Question 53

What are the steps for Mass spectrometry-based proteomics?

Answer 53

1. Extract proteins from biological sample
2. Trypsin digestion into peptides
3. Separate proteins from each other or enrich proteins with specific PTM
4. Determine idetities of proteins
   
   • Obtain partial sequences by MS
   • Identify full sequences from databases
5. Determine quantities of proteins
6. Verify post-translational modifications

Question 54

Definition

a RNA-binding protein which binds to the poly(A) tail of mRNA

Answer 54

Poly-A-binding protein

Question 55

What questions do we ask about tyrosines in a chemokine protein?

Answer 55

Are they sulfated?

Are they important for funciton?

Is the sulfation important for function?

Question 56

Definition

the chemical addition of a phosphoryl group (PO3-) to an organic molecule

Answer 56

Phosphorylation

Question 57

Definition

an endopeptidase that breaks down proteins into smaller peptides (that is, a protease). It is produced in the stomach and is one of the main digestive enzymes in the digestive systems of humans and many other animals, where it helps digest the proteins in food.

Answer 57

Pepsin

Question 58

At which of the following denaturant concentrations is the protein in the native state?

Select one:

1. 5 M
2. 5 M

4 M

6 M

Answer 58

At which of the following denaturant concentrations is the protein in the native state?

Select one:

1.5 M

3.5 M

4 M

6 M

Question 59

Why is phenylalanine often used in studies of tyrosine sulfation?

Answer 59

Phenylalanine is very structurall different to tyrosine but it cannot be sulfated since it doesn’t have a alcohol group

Question 60

Definition

the third and final binding site for t-RNA in the ribosome during protein synthesis

Answer 60

E site (exit)

Question 61

_________ evolution often results in proteins with similar folds which share no significant sequence identity

Answer 61

Convergent evolution often results in proteins with similar folds which share no significant sequence identity

Question 62

What does Lane 1 and Lane 2 tell us?

Answer 62

Lane 1 = no band

Lane 2 = band present (CCR5)

Tells us that the immunoprecipitation worked (i.e. this was the control comparison for CCR5)

In the absence of transfection, CCR5 is not expressed

Question 63

Define

Transition state

Answer 63

a very short-lived configuration of atoms at a local energy maximum in a reaction-energy diagram

Question 64

Definition

the covalent and generally enzymatic modification of proteins following protein biosynthesis

Answer 64

Post-translational modifications

Question 65

According to transition state theory, the relation between the first-order rate constant and the activation energy is _________ and _________

Answer 65

According to transition state theory, the relation between the first-order rate constant and the activation energy is inverse and exponential

Question 66

Which of these proteins would be phosphorylated?

Answer 66

Orange

More negatively charged = lower pI

Question 67

What does elongation consist of?

Answer 67

1. Binding of tRNA 2. Peptide bond formation 3. Translocation

Question 68

Answer 68

Question 69

What does comparing Lane 6 and Lane 2 tell us?

Answer 69

Tells us that CCR5 is sulfated

Question 70

What are enzymes?

Answer 70

Enzymes are proteins that catalyse biochemical reactions

Question 71

Definition

a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins

Answer 71

Trypsin

Question 72

What factors influence protein levels?

Answer 72

• Localisation
  
  • Subcellular organelles
  • Multiprotein complexes
  • Membranes
  • Extracellular spaces
• Posttranslational Modifications (PTM)
  
  • Proteolytic cleavage
  • Acetylation
  • Methylation
  • Phosphorylation
• Damage and/or degradation

Question 73

How many beta sheets do RNA-recognition motifs have?

Answer 73

At least 4

Question 74

Definition

antigen that is located on the surface of red blood cells, and is a glycosylated membrane protein and non-specific receptor for several chemokines. The protein is also the receptor for the human malarial parasites

Answer 74

Duffy Antigen and Receptor for Chemokines (DARC)

Question 75

Define

Zymogen

Answer 75

an inactive substance which is converted into an enzyme when activated by another enzyme

Question 76

Define

Tyrosine sulfation

Answer 76

a posttranslational modification where a sulfate group is added to a tyrosine residue of a protein molecule

Question 77

Which amino acids contain sulfur?

Answer 77

Cys and Met

Question 78

Definition

an inactive substance which is converted into an enzyme when activated by another enzyme

Answer 78

Zymogen

Question 79

What does the Duffy antigen do?

Answer 79

Determines blood type (A/B/O)

Question 80

What is the role of the hydrophobic pocket of chymotrypsin?

Answer 80

When substrate binds, the side chain of the residue adjacent to the peptide bond to be cleaved nestles in a hydrophobic pocket on the enzyme, positioning the peptide for attack

Question 81

How do enzymes work?

Answer 81

• Enzymes can bring reactants close together
• Enzymes stabilise the transition state of a reaction, thus lowering the activation energy required for the reaction

Question 82

Define

Expressional proteomics

Answer 82

a branch of proteomics that includes the analysis of protein expression at larger scale. Focused on studying complete proteomes, e.g. from two differentially treated cell lines

Question 83

How many aromatic residues does the RNA-recognition motif have?

Answer 83

3 (tyr or phe)

Question 84

Amino acid sequence identity of ~__% strongly suggests proteins have same fold

Answer 84

Amino acid sequence identity of ~30% strongly suggests proteins have same fold

Question 85

Define

Chaperone

Answer 85

proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures

Question 86

Definition

a structurally conserved protein domain contained within the Src oncoprotein and in many other intracellular signal-transducing proteins. They allow proteins containing those domains to dock to phosphorylated tyrosine residues on other proteins

Answer 86

SH2 domain

Question 87

What questions does Expressional proteomics ask?

Answer 87

What proteins are present?

What are their expression levels?

What are their PTMs?

Question 88

What is characteristic of the chemokine levels of individuals with HIV resistance?

Answer 88

Individuals with HIV resistance have higher levels of chemokines. Chemokines block CCR5 so HIV cannot bind

Question 89

Where does tyrosine sulfation occur?

Answer 89

Present in many secreted and cell surface proteins. Occurs in the trans-Golgi network

Question 90

The four residues indicated with asterisks (*) are found in the same positions in every chemokine. Which of the following is the likely role of these conserved residues?

Select one:

Forming hydrophobic interactions within the protein core

Forming salt bridges (ionic interactions) within the protein core

Forming salt bridges (ionic interactions) with residues in the corresponding receptors

Forming disulfide bonds

Forming one or more loop structure(s)

Answer 90

The four residues indicated with asterisks (*) are found in the same positions in every chemokine. Which of the following is the likely role of these conserved residues?

Select one:

Forming hydrophobic interactions within the protein core

Forming salt bridges (ionic interactions) within the protein core

Forming salt bridges (ionic interactions) with residues in the corresponding receptors

Forming disulfide bonds

Forming one or more loop structure(s)

Question 91

Which Tyr residues in the N-terminus of DARC are sulfated?

Answer 91

Both 30 and 41

Question 92

What does initiation consist of?

Answer 92

1. mRNA binding 2. Initiation tRNA binding 3. Ribosome assembly

Question 93

Define

Trypsin

Answer 93

a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins

Question 94

What are the three main steps in translation?

Answer 94

Initiation Elongation Termination

Question 95

How do enzymes accelerate biochemical reactions?

Answer 95

By lowering the activation energy of a reaction

Question 96

What is more conserved in proteins: sequence or structure?

Answer 96

Structure

Question 97

Define

SH2 domain

Answer 97

a structurally conserved protein domain contained within the Src oncoprotein and in many other intracellular signal-transducing proteins. They allow proteins containing those domains to dock to phosphorylated tyrosine residues on other proteins

Question 98

Definition

a substance (other than the substrate) whose presence is essential for the activity of an enzyme

Answer 98

Cofactor

Question 99

This curve indicates that…

Select one:

a single interaction in the native structure contributes to the low Gibbs free energy of the protein.

the native state is the most stable state.

interactions that stabilise the native state are cooperative with each other.

the protein is unfolded in the absence of denaturing agent.

Answer 99

This curve indicates that…

Select one:

a single interaction in the native structure contributes to the low Gibbs free energy of the protein.

the native state is the most stable state.

interactions that stabilise the native state are cooperative with each other.

the protein is unfolded in the absence of denaturing agent.

Question 100

Definition

a molecule or substance that has a tendency to donate electrons or react at electron-poor sites such as protons.

Answer 100

Nucleophile

Question 101

Define

Coenzyme

Answer 101

a non-protein compound that is necessary for the functioning of an enzyme

Question 102

What are the cleavage points for chymotrypsin?

Answer 102

Phe, Trp and Tyr

Question 103

How many different types of tRNAs are required for recognising 61 codons?

Answer 103

31 + 1(for fMet-tRNA) = 32

Question 104

How can proteomics improve cancer treatment?

Answer 104

Enables personalised medicine - can effectively narrow down the best treatment to target individual diagnosis

Individual tumours could be characterised to determine which proteins have been up/downregulated. Potentially reducing side-effects

Question 105

What type of PTM can induce protein degradation?

Answer 105

Polyubiquitylation

Question 106

You have determined the crystal structure of an enzyme in complex with the product of the reaction. You have mutated each of the residues lining the ligand binding site to alanine and measured the activity of the variant enzyme. The activity of the His34Ala variant was 1,000 times less than the activity of the native enzyme. The activities of Ser17Ala and Tyr100Ala variants were reduced 6 fold and 15 fold in comparison to the native enzyme. Which is the best conclusion that can be drawn from this observation?

Select one:

All three residues are directly involved in the catalysis.

All three residues mediate catalysis, but His34 catalyses the first step, whereas Ser17 and Tyr100 catalyse the second step.

His34 is a catalytic residue, whilst Ser17 and Tyr100 likely form direct stabilising interactions with the substrate, but are not involved in catalysis.

His34 and Tyr100 are catalytic residues, whilst Ser17 likely forms direct stabilising interactions with the substrate, but is not involved in catalysis.

His34 is in the enzyme’s active site, whilst Ser17 and Tyr100 definitely do not.

Answer 106

You have determined the crystal structure of an enzyme in complex with the product of the reaction. You have mutated each of the residues lining the ligand binding site to alanine and measured the activity of the variant enzyme. The activity of the His34Ala variant was 1,000 times less than the activity of the native enzyme. The activities of Ser17Ala and Tyr100Ala variants were reduced 6 fold and 15 fold in comparison to the native enzyme. Which is the best conclusion that can be drawn from this observation?

Select one:

All three residues are directly involved in the catalysis.

All three residues mediate catalysis, but His34 catalyses the first step, whereas Ser17 and Tyr100 catalyse the second step.

His34 is a catalytic residue, whilst Ser17 and Tyr100 likely form direct stabilising interactions with the substrate, but are not involved in catalysis.

His34 and Tyr100 are catalytic residues, whilst Ser17 likely forms direct stabilising interactions with the substrate, but is not involved in catalysis.

His34 is in the enzyme’s active site, whilst Ser17 and Tyr100 definitely do not.

Question 107

Define

P site (peptidyl)

Answer 107

the second binding site for tRNA in the ribosome

Question 108

Which regions of the Poly-A-binding protien have been conserved?

Answer 108

1. residues in the hydrophobic tract
2. Residues that recognise poly-A

Question 109

Define

RNA-recognition motif

Answer 109

a putative RNA-binding domain of about 90 amino acids that are known to bind single-stranded RNAs

Question 110

Define

A site (aminoacyl)

Answer 110

the first binding site in the ribosome

Question 111

A protein domain is…

Select one:

an autonomously folded structural unit of a protein.

a type of structure that is present in all proteins.

the part of a protein that catalyses reactions.

an unstructured region in a protein.

the region of a protein with the most disulfide bonds.

Answer 111

A protein domain is…

Select one:

an autonomously folded structural unit of a protein.

a type of structure that is present in all proteins.

the part of a protein that catalyses reactions.

an unstructured region in a protein.

the region of a protein with the most disulfide bonds.

Question 112

Define

Post-translational modifications

Answer 112

the covalent and generally enzymatic modification of proteins following protein biosynthesis

Question 113

Definition

a posttranslational modification where a sulfate group is added to a tyrosine residue of a protein molecule

Answer 113

Tyrosine sulfation

Question 114

Definition

enzymes that catalyze post-translational tyrosine sulfation of proteins

Answer 114

Tyrosylprotein Sulfotransferases (TPSTs)

Question 115

Define

Cofactor

Answer 115

a substance (other than the substrate) whose presence is essential for the activity of an enzyme

Question 116

What does the addition of urea do to a protein?

Answer 116

Denatures it by forming hydrogen bonds with the proteins that disrupted the hydrogen bonds between the amino acids

Question 117

What does Lane 3 and Lane 4 tell us?

Answer 117

Lane 3 = no band

Lane 4 = band present (CD4)

Tells us that the immunoprecipitation worked (i.e. this was the control comparison for CD4)

In the absence of transfection, CD4 is not expressed

Question 118

What are the 2 distinct stages of the action of Chymotrypsin?

Answer 118

1. Formation of acyl-enzyme intermediate
2. Hydrolysis of acyl-enzyme intermediate

Question 119

What do inflammation, HIV and Malaria all have in common?

Answer 119

They all involve chemokine receptors

Question 120

What are some situations in which the Thermodynamic Hypothesis is not valid?

Answer 120

Some proteins aggregate under certain conditions In vivo folding may require chaperone proteins Zymogens: Fold, then are cleaved, mature proteins may be “trapped” in zymogen-like fold Some proteins have more than one “native” structure

Question 121

Definition

a branch of proteomics that includes the analysis of protein expression at larger scale. Focused on studying complete proteomes, e.g. from two differentially treated cell lines

Answer 121

Expressional proteomics

Question 122

What does comparing Lane 8 and Lane 4 tell us?

Answer 122

Shows that CD4 is not sulfated

Immunoprecipitation worked due to lane 4 band

Question 123

True or False:

Like phosphorylation, sulfation is reversible

Answer 123

False

Phosphorylation is reversible but sulfation is not.

Question 124

What is a protein domain?

Answer 124

A protein domain is a spatially distinct structural component that could conceivably fold and function in isolation

Question 125

Why is chymotrypsin a member of the serine protease family?

Answer 125

Ser is the key active site residue

Question 126

True or False: Sequences of low similarity can have the same 3D structure

Answer 126

True

Question 127

Define

Reaction intermediate

Answer 127

a molecular entity that is formed from the reactants (or preceding intermediates) and reacts further to give the directly observed products of a chemical reaction

Question 128

What does the SH2 domain do?

Answer 128

The SH2 domain has evolved to recognise phosphotyrosines

Question 129

What conclusions can be draw based in this figure?

Answer 129

Both tyrosines are important but tyrosine 14 is more important to chemokine response

Question 130

Define

Proteome

Answer 130

a set of proteins produced in an organism, system or biological context

Question 131

True or False:

The transition state of a compound is stabile and can be crystallised

Answer 131

False

Transition state is not a stable chemical species, it has no lifetime

Question 132

Tyrosylprotein Sulfotransferases are selective for tyr in what kind of environment?

Answer 132

Acidic (aspartic and glutamic acid)

Question 133

Define

Tyrosylprotein Sulfotransferases (TPSTs)

Answer 133

enzymes that catalyze post-translational tyrosine sulfation of proteins

Question 134

Describe the role of NAD+ in redox reactions

Answer 134

In a redox reaction, 2 electrons and a proton are transferred to NAD+, forming NADH

Question 135

What two types of ions are formed when peptides are broken for mass spec? Which ion produces peaks?

Answer 135

b-type ions

y-type ions (produce peaks)

Question 136

Answer 136

Red

Question 137

Definition

the first binding site in the ribosome

Answer 137

A site (aminoacyl)

Question 138

Define

Electrophile

Answer 138

positively charged or neutral species having vacant orbitals that are attracted to an electron rich centre

Question 139

Which features of proteins are used for 2D Gel electrophoresis?

Answer 139

Isoelectric focusing (pI)

Size

Question 140

What type of receptors are needed for HIV to fuse with target cells?

Answer 140

Chemokine receptors

Question 141

What are some characteristics of enzymes?

Answer 141

1. Enzymes work very rapidly
2. Activity dependent on pH
3. Activity dependent on temperature
4. Enzymes are substrate specific
5. Many enzymes require cofactors or coenzymes
6. Enzymes can be inhibited

Question 142

Definition

proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures

Answer 142

Chaperone

Question 143

Define

E site (exit)

Answer 143

the third and final binding site for t-RNA in the ribosome during protein synthesis

Question 144

Which of the following conclusions can be drawn from these data?

Select one:

Most of the proteins are more stable at higher temperature

The mutant protein is more stable than the wild-type protein

Most proteins are more stable at lower temperature

At 60oC the wild-type protein is mostly folded but the mutant protein is mostly unfolded

At 60oC both proteins are fully folded

Answer 144

Which of the following conclusions can be drawn from these data?

Select one:

Most of the proteins are more stable at higher temperature

The mutant protein is more stable than the wild-type protein

Most proteins are more stable at lower temperature

At 60oC the wild-type protein is mostly folded but the mutant protein is mostly unfolded

At 60oC both proteins are fully folded

Question 145

Define

Zinc finger domains

Answer 145

a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) in order to stabilize the fold

Question 146

What are the cleavage points for pepsin?

Answer 146

Leu, Phe, Trp and Tyr

Question 147

Chymotrypsin is a well studied serine protease. It has evolved to…

Select one:

cleave a peptide bond that is C-terminal to a serine residue.

stabilise an acyl-enzyme intermediate in the binding site through hydrogen bonding interactions.

cleave a peptide bond that is N-terminal to an aromatic residue.

catalyse the direct hydrolysis of a peptide bond by positioning a water molecule close to the peptide bond.

catalyse the cleavage of trypsin to form trypsinogen.

Answer 147

Chymotrypsin is a well studied serine protease. It has evolved to…

Select one:

cleave a peptide bond that is C-terminal to a serine residue.

stabilise an acyl-enzyme intermediate in the binding site through hydrogen bonding interactions.

cleave a peptide bond that is N-terminal to an aromatic residue.

catalyse the direct hydrolysis of a peptide bond by positioning a water molecule close to the peptide bond.

catalyse the cleavage of trypsin to form trypsinogen.

Question 148

Define

Proteomics

Answer 148

a branch of biotechnology concerned with applying the techniques of molecular biology, biochemistry, and genetics to analyzing the structure, function, and interactions of the proteins produced by the genes of a particular cell, tissue, or organism, with organizing the information in databases, and with applications of the data

Question 149

What does termination consist of?

Answer 149

1. Release of polypeptide 2. Ribosome disassembly

Question 150

Which of the following statements about the active site of an enzyme is correct?

Select one:

The active site of an enzyme binds the substrate of the reaction it catalyses more tightly than the transition state.

The active site of an enzyme binds the substrate of the reaction it catalyses less tightly than the transition state.

The active site of an enzyme binds the product of the reaction it catalyses more tightly than the transition state.

The active site of an enzyme makes better contacts with the substrate of the reaction it catalyses than with the transition state.

The active site of an enzyme binds the product of the reaction it catalyses more tightly than the substrate.

Answer 150

Which of the following statements about the active site of an enzyme is correct?

Select one:

The active site of an enzyme binds the substrate of the reaction it catalyses more tightly than the transition state.

The active site of an enzyme binds the substrate of the reaction it catalyses less tightly than the transition state.

The active site of an enzyme binds the product of the reaction it catalyses more tightly than the transition state.

The active site of an enzyme makes better contacts with the substrate of the reaction it catalyses than with the transition state.

The active site of an enzyme binds the product of the reaction it catalyses more tightly than the substrate.

Question 151

Define

G-protein-coupled receptors

Answer 151

a large protein family of receptors that detect molecules outside the cell and activate internal signal transduction pathways and, ultimately, cellular responses

Question 152

How many transmembrane regions do chemokine receptors have?

Answer 152

7

Question 153

Define

Duffy Antigen and Receptor for Chemokines (DARC)

Answer 153

antigen that is located on the surface of red blood cells, and is a glycosylated membrane protein and non-specific receptor for several chemokines. The protein is also the receptor for the human malarial parasites

Question 154

Which amino acids does tyrosine need to be near to be sulfated?

Answer 154

Aspartic acid

Glutamic acid

Question 155

How many nucleotides make up a codon?

Answer 155

3

Question 156

Definition

the second binding site for tRNA in the ribosome

Answer 156

P site (peptidyl)

Question 157

Definition

a set of proteins produced in an organism, system or biological context

Answer 157

Proteome

Question 158

Which cells incorporate ³⁵S-Cys/Met? Which cells incorporate ³⁵S-SO₄^2-?

Answer 158

All cells incorporate ³⁵S-Cys/Met

³⁵S-SO₄^2- only incorporated if cell contains sulfate

Question 159

True or False:

Enzymes are protein catalyst

Answer 159

False

They are biological catalysts

Most are protein but some are RNA (i.e. proteosome)

Question 160

Which PTM occurs on tyrosine residues and adds negative charges to the protein?

a) Methylation
b) Ubiquitination
c) Phosphorylation

Answer 160

Which PTM occurs on tyrosine residues and adds negative charges to the protein?

a) Methylation
b) Ubiquitination

c) Phosphorylation

Question 161

What are the cleavage points for trypsin?

Answer 161

Lys and Arg

Question 162

Definition

the amino acids directly involved in chemical catalysis. They mainly act as a general acid–base, electrophilic or nucleophilic catalyst or they polarize and stabilize the transition state

Answer 162

Catalytic residues

Question 163

How are some people resistant to HIV?

Answer 163

Mutation of the CCR5 chemokine receptor so that HIV cannot fuse with the target cell effectively

Question 164

Definition

a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) in order to stabilize the fold

Answer 164

Zinc finger domains

Question 165

Knockout of mouse orthologues of Tyrosylprotein Sulfotransferase causes what?

Answer 165

• Reduced body weight
• Reduced fertility
• Early death (in double knockout)

Question 166

Define

Pepsin

Answer 166

an endopeptidase that breaks down proteins into smaller peptides (that is, a protease). It is produced in the stomach and is one of the main digestive enzymes in the digestive systems of humans and many other animals, where it helps digest the proteins in food.

Question 167

Definition

the 3rd nucleotide in a codon. Binding of a codon in an mRNA the cognate tRNA is much “looser” in the third position of the codon

Answer 167

Wobble position

Question 168

In the section of the 2D gel shown below, the proteins were separated first by isoelectric focusing (moving from left to right) and second by SDS-PAGE (moving from top to bottom). The proteins were each identified by a specific antibody to be phosphorylated cofilin.

What is the most likely difference between protein “c1” and protein “c5”?

Select one:

c5 is less phosphorylated than c1

c5 has a lower molecular weight than c1

c5 has fewer disulphide bonds than c1

c5 has a lower isoelectric point than c1

Answer 168

What is the most likely difference between protein “c1” and protein “c5”?

Select one:

c5 is less phosphorylated than c1

c5 has a lower molecular weight than c1

c5 has fewer disulphide bonds than c1

c5 has a lower isoelectric point than c1

Question 169

When a sample of a protein is treated with increasing concentrations of a denaturing agent, the following unfolding curve is obtained.

At which of the following denaturant concentrations does the protein have the highest Gibbs free energy?

Select one:

1. 5 M
2. 5 M
3. 5 M

6 M

Answer 169

At which of the following denaturant concentrations does the protein have the highest Gibbs free energy?

Select one:

1. 5 M
2. 5 M
3. 5 M

6 M

Question 170

In stomach tumours, protein X is present in a phosphorylated form in which a tyrosine residue near the C-terminus is phosphorylated. You are given the cell lysate sample of a stomach cancer patient and are asked to determine if it contains the phosphorylated form of protein X. Which of the following procedures should you use?

Select one:

Digestion with trypsin → immunoprecipitation using an antibody that specifically recognizes phosphorylated tyrosine → 2D gel electrophoresis

Digestion with trypsin → immunoprecipitation using an antibody that specifically recognizes phosphorylated tyrosine → tandem mass spectrometry

Isoelectric focusing → 2D gel electrophoresis → immunoprecipitation using an antibody that specifically recognizes phosphorylated tyrosine

BLAST search → 1D gel electrophoresis → 2D gel electrophoresis

Answer 170

In stomach tumours, protein X is present in a phosphorylated form in which a tyrosine residue near the C-terminus is phosphorylated. You are given the cell lysate sample of a stomach cancer patient and are asked to determine if it contains the phosphorylated form of protein X. Which of the following procedures should you use?

Select one:

Digestion with trypsin → immunoprecipitation using an antibody that specifically recognizes phosphorylated tyrosine → 2D gel electrophoresis

Digestion with trypsin → immunoprecipitation using an antibody that specifically recognizes phosphorylated tyrosine → tandem mass spectrometry

Isoelectric focusing → 2D gel electrophoresis → immunoprecipitation using an antibody that specifically recognizes phosphorylated tyrosine

BLAST search → 1D gel electrophoresis → 2D gel electrophoresis

Question 171

Which of the following statements about enzymes is FALSE?

Select one:

Enzymes speed up a chemical reaction

The active site on the enzyme may undergo a slight change in shape when the substrate binds

Enzymes are very specific in their action

The presence of an enzyme will change the outcome of a reaction

Answer 171

Which of the following statements about enzymes is FALSE?

Select one:

Enzymes speed up a chemical reaction

The active site on the enzyme may undergo a slight change in shape when the substrate binds

Enzymes are very specific in their action

The presence of an enzyme will change the outcome of a reaction

Question 172

The major conclusion from the Anfinsen experiment was…

Select one:

mercaptoethanol is able to disrupt secondary structures but unable to break up disulfide bonds

the native 3D structure of a protein is defined by its amino acid sequence

disulfide bonds are required for proteins to be correctly folded

all proteins fold spontaneously to their lowest energy structures

Answer 172

The major conclusion from the Anfinsen experiment was…

Select one:

mercaptoethanol is able to disrupt secondary structures but unable to break up disulfide bonds

the native 3D structure of a protein is defined by its amino acid sequence

disulfide bonds are required for proteins to be correctly folded

all proteins fold spontaneously to their lowest energy structures

Question 173

The following figures describe two consequent steps in the reaction catalysed by chymotrypsin.

Fig. 1 shows…

Select one:

transition state of the reaction

complex of chymotrypsin with the product of the reaction

covalent acyl-enzyme intermediate that is not very stable

nucleophilic attack of the peptide carbonyl group by Ser195

Answer 173

Fig. 1 shows…

Select one:

transition state of the reaction

complex of chymotrypsin with the product of the reaction

covalent acyl-enzyme intermediate that is not very stable

nucleophilic attack of the peptide carbonyl group by Ser195

Question 174

The following figures describe two consequent steps in the reaction catalysed by chymotrypsin.

Substitution of His57 with Ala would result in…

Select one:

loss of the ion bridge between Ser195 and His57

~10-fold reduction in the enzyme activity

>1000-fold reduction in the enzyme activity

specificity towards sugars rather than proteins

Answer 174

Substitution of His57 with Ala would result in…

Select one:

loss of the ion bridge between Ser195 and His57

~10-fold reduction in the enzyme activity

>1000-fold reduction in the enzyme activity

specificity towards sugars rather than proteins

Question 175

The following figures describe two consequent steps in the reaction catalysed by chymotrypsin.

Fig. 2 shows…

Select one:

transition state

enzyme-product complex

covalent acyl-enzyme intermediate

nucleophilic attack by Ser195

Answer 175

Fig. 2 shows…

Select one:

transition state

enzyme-product complex

covalent acyl-enzyme intermediate

nucleophilic attack by Ser195

Question 176

Below is an alignment of a region of the hemoglobin sequence from various animal species.

From the sequence similarity seen between these sequences, which of the following conclusions is supported?

Select one:

that humans are more closely related to alligators than rats

that these protein sequences possess similar three-dimensional structures

that these proteins are likely to have different functions from each other

that the domestic duck and Canadian goose are the same species

Answer 176

From the sequence similarity seen between these sequences, which of the following conclusions is supported?

Select one:

that humans are more closely related to alligators than rats

that these protein sequences possess similar three-dimensional structures

that these proteins are likely to have different functions from each other

that the domestic duck and Canadian goose are the same species

Question 177

Below is an alignment of a region of the hemoglobin sequence from various animal species.

The residues indicated with a full stop (.) indicate amino acid residues that…

Select one:

are identical in each of these protein sequences

have side chains with highly similar properties

have side chains with a degree of similarity

are highly variable in each of these protein sequences

Answer 177

The residues indicated with a full stop (.) indicate amino acid residues that…

Select one:

are identical in each of these protein sequences

have side chains with highly similar properties

have side chains with a degree of similarity

are highly variable in each of these protein sequences

Question 178

The presence of intense bands at ~38 kDa in lane 2 AND at ~54 kDa in lane 4 but no corresponding bands in lanes 1 and 3 indicates that…

Select one:

The cells express CD4 even without being transfected with the expression vector

The cells express CCR5 even without being transfected with the expression vector

The cells express CD4 only after transfection with the expression vector

The cells express both CD4 and CCR5 only after transfection with the expression vector

Answer 178

The presence of intense bands at ~38 kDa in lane 2 AND at ~54 kDa in lane 4 but no corresponding bands in lanes 1 and 3 indicates that…

Select one:

The cells express CD4 even without being transfected with the expression vector

The cells express CCR5 even without being transfected with the expression vector

The cells express CD4 only after transfection with the expression vector

The cells express both CD4 and CCR5 only after transfection with the expression vector

Question 179

Lane 7 does NOT show any intense bands because…

Select one:

Sulfate blocks the expression of CD4 by non-transfected cells

Non-transfected cells do not express CD4

Transfected cells do not express CCR5 in the presence of sulfate

Transfected cells do not express CD4 in the presence of sulfate

Answer 179

Lane 7 does NOT show any intense bands because…

Select one:

Sulfate blocks the expression of CD4 by non-transfected cells

Non-transfected cells do not express CD4

Transfected cells do not express CCR5 in the presence of sulfate

Transfected cells do not express CD4 in the presence of sulfate

Question 180

Keeping in mind the results in lanes 1-4, the absence of any intense bands in lane 8 indicates that…

Select one:

CCR5 prevents the sulfation of CD4

The antibody used is unable to efficiently precipitate CD4

CCR5 is not sulfated

CD4 is not sulfated

Answer 180

Keeping in mind the results in lanes 1-4, the absence of any intense bands in lane 8 indicates that…

Select one:

CCR5 prevents the sulfation of CD4

The antibody used is unable to efficiently precipitate CD4

CCR5 is not sulfated

CD4 is not sulfated