Protein Expression & Modification Flashcards
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
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
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
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
Definition
positively charged or neutral species having vacant orbitals that are attracted to an electron rich centre
Electrophile
Sulfation ________ binding to chemokines and to pathogen proteins
Sulfation enhances binding to chemokines and to pathogen proteins
Define
Poly-A-binding protein
a RNA-binding protein which binds to the poly(A) tail of mRNA
What are the three types of proteomics?
Expressional proteomics
Functional proteomics
Structural proteomics
Define
Wobble position
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
Definition
a putative RNA-binding domain of about 90 amino acids that are known to bind single-stranded RNAs
RNA-recognition motif
Why is proteomics important?
- To obtain the most complete possible information about the components of cells
- To determine the presence and effects of protein modifications
- Systems biology - a “holistic” view of the network of interactions leading to biological phenotypes
- Identification of cell-type markers
- Identification of disease markers
- Identification of potential drug targets
- Tracking the progress of disease or therapy
Why do we need to identify phosphorylated proteins in the cell?
Phosphorylation cascades are involved in many signalling pathways
What two things can mass spec identify in proteins?
- Determine mass of protein
- Sequence peptides
Which type of PTM introduces a negatively charge and hyrophilic group?
Phosphorylation
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
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
True or False:
The same regio that bonds chemokines also binds malaria
True
Which position is denoted the “wobble position”?
Third nucleotide in a codon
What PTMs do chemokine receptors have?
Tyrosine sulfation
N-linked glycosylation
Phosphorylation
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)
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)
Definition
a very short-lived configuration of atoms at a local energy maximum in a reaction-energy diagram
Transition state
Why does the enzyme are the product dissociate?
They have low affinity for one another
True or False:
PTMs can both enhance or inhibit protein function
True
Define
Nucleophile
a molecule or substance that has a tendency to donate electrons or react at electron-poor sites such as protons.
What does Tyrosylprotein Sulfotransferases catalyse?
Catalyse transfer of sulfate from PAPS to tyr
Definition
a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides
Chymotrypsin
Which regions of chemokine receptors are sulfated?
N-terminus
Inhibitors that mimic the reaction transition state show very strong/weak binding to the enzymes
Inhibitors that mimic the reaction transition state show very strong binding to the enzymes
Definition
a non-protein compound that is necessary for the functioning of an enzyme
Coenzyme
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?
Although powdered glucose is highly flammable, it requires input of activation energy in form of heat (i.e. the energy barrier is high)
Why does the native structure of a protein form spontaneously?
The native structure is thermodynamically favoured. i.e. it has the lowest Gibbs free energy
Define
Phosphorylation
the chemical addition of a phosphoryl group (PO3-) to an organic molecule
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)
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)
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
Reaction intermediate
What is Functional proteomics?
Studies more limited protein sets to determine their function
e.g. identify interacting proteins in vivo
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
Proteomics
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
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
Define
Catalytic residues
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
Why can we not deduce anything about sulfation based on Lanes 1-4?
We cannot deduce anything about sulfation since they were labelled non-specifically (i.e. with 35S-Cys/Met)
What does the addition of Mercaptoethanol (BME) do to a protein?
Denatures it by breaking disulphide bonds
Which features of proteins are used for 1D Gel electrophoresis?
Size
Definition
a large protein family of receptors that detect molecules outside the cell and activate internal signal transduction pathways and, ultimately, cellular responses
G-protein-coupled receptors
Is the Thermodynamic Hypothesis valid for proteins without disulphide bonds?
Yes
What recognises stop codons?
Release factors
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?
The lowest
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
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
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
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
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.
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.
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
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
Chymotrypsin catalyses the cleavage of peptide bonds on which terminal of which amino acids?
Cleavage of peptide bonds on the carboxy-terminal side of Phe, Trp and Tyr residues
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
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
What are the steps to 2D Gel Electrophoresis?
- Separation accoridng to pI (charge)
- Proteins travel until neutrally charged
- Separation according to molecular weight
Define
Chymotrypsin
a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides
Which part of the RNA-recognition motif binds to RNA?
The face of the beta sheet
What are the steps for Mass spectrometry-based proteomics?
- Extract proteins from biological sample
- Trypsin digestion into peptides
- Separate proteins from each other or enrich proteins with specific PTM
- Determine idetities of proteins
- Obtain partial sequences by MS
- Identify full sequences from databases
- Determine quantities of proteins
- Verify post-translational modifications
Definition
a RNA-binding protein which binds to the poly(A) tail of mRNA
Poly-A-binding protein
What questions do we ask about tyrosines in a chemokine protein?
Are they sulfated?
Are they important for funciton?
Is the sulfation important for function?
Definition
the chemical addition of a phosphoryl group (PO3-) to an organic molecule
Phosphorylation
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.
Pepsin
At which of the following denaturant concentrations is the protein in the native state?
Select one:
- 5 M
- 5 M
4 M
6 M
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
Why is phenylalanine often used in studies of tyrosine sulfation?
Phenylalanine is very structurall different to tyrosine but it cannot be sulfated since it doesn’t have a alcohol group
Definition
the third and final binding site for t-RNA in the ribosome during protein synthesis
E site (exit)
_________ evolution often results in proteins with similar folds which share no significant sequence identity
Convergent evolution often results in proteins with similar folds which share no significant sequence identity
What does Lane 1 and Lane 2 tell us?
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
Define
Transition state
a very short-lived configuration of atoms at a local energy maximum in a reaction-energy diagram
Definition
the covalent and generally enzymatic modification of proteins following protein biosynthesis
Post-translational modifications
According to transition state theory, the relation between the first-order rate constant and the activation energy is _________ and _________
According to transition state theory, the relation between the first-order rate constant and the activation energy is inverse and exponential
Which of these proteins would be phosphorylated?
Orange
More negatively charged = lower pI
What does elongation consist of?
- Binding of tRNA 2. Peptide bond formation 3. Translocation
What does comparing Lane 6 and Lane 2 tell us?
Tells us that CCR5 is sulfated
What are enzymes?
Enzymes are proteins that catalyse biochemical reactions
Definition
a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins
Trypsin
What factors influence protein levels?
- Localisation
- Subcellular organelles
- Multiprotein complexes
- Membranes
- Extracellular spaces
- Posttranslational Modifications (PTM)
- Proteolytic cleavage
- Acetylation
- Methylation
- Phosphorylation
- Damage and/or degradation
How many beta sheets do RNA-recognition motifs have?
At least 4
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
Duffy Antigen and Receptor for Chemokines (DARC)
Define
Zymogen
an inactive substance which is converted into an enzyme when activated by another enzyme
Define
Tyrosine sulfation
a posttranslational modification where a sulfate group is added to a tyrosine residue of a protein molecule
Which amino acids contain sulfur?
Cys and Met
Definition
an inactive substance which is converted into an enzyme when activated by another enzyme
Zymogen
What does the Duffy antigen do?
Determines blood type (A/B/O)
What is the role of the hydrophobic pocket of chymotrypsin?
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
How do enzymes work?
- Enzymes can bring reactants close together
- Enzymes stabilise the transition state of a reaction, thus lowering the activation energy required for the reaction
Define
Expressional proteomics
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
How many aromatic residues does the RNA-recognition motif have?
3 (tyr or phe)
Amino acid sequence identity of ~__% strongly suggests proteins have same fold
Amino acid sequence identity of ~30% strongly suggests proteins have same fold
Define
Chaperone
proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures
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
SH2 domain
What questions does Expressional proteomics ask?
What proteins are present?
What are their expression levels?
What are their PTMs?
What is characteristic of the chemokine levels of individuals with HIV resistance?
Individuals with HIV resistance have higher levels of chemokines. Chemokines block CCR5 so HIV cannot bind
Where does tyrosine sulfation occur?
Present in many secreted and cell surface proteins. Occurs in the trans-Golgi network
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)
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)
Which Tyr residues in the N-terminus of DARC are sulfated?
Both 30 and 41
What does initiation consist of?
- mRNA binding 2. Initiation tRNA binding 3. Ribosome assembly
Define
Trypsin
a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins
What are the three main steps in translation?
Initiation Elongation Termination
How do enzymes accelerate biochemical reactions?
By lowering the activation energy of a reaction
What is more conserved in proteins: sequence or structure?
Structure
Define
SH2 domain
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
Definition
a substance (other than the substrate) whose presence is essential for the activity of an enzyme
Cofactor
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.
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.
Definition
a molecule or substance that has a tendency to donate electrons or react at electron-poor sites such as protons.
Nucleophile
Define
Coenzyme
a non-protein compound that is necessary for the functioning of an enzyme
What are the cleavage points for chymotrypsin?
Phe, Trp and Tyr
How many different types of tRNAs are required for recognising 61 codons?
31 + 1(for fMet-tRNA) = 32
How can proteomics improve cancer treatment?
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
What type of PTM can induce protein degradation?
Polyubiquitylation
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.
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.
Define
P site (peptidyl)
the second binding site for tRNA in the ribosome
Which regions of the Poly-A-binding protien have been conserved?
- residues in the hydrophobic tract
- Residues that recognise poly-A
Define
RNA-recognition motif
a putative RNA-binding domain of about 90 amino acids that are known to bind single-stranded RNAs
Define
A site (aminoacyl)
the first binding site in the ribosome
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.
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.
Define
Post-translational modifications
the covalent and generally enzymatic modification of proteins following protein biosynthesis
Definition
a posttranslational modification where a sulfate group is added to a tyrosine residue of a protein molecule
Tyrosine sulfation
Definition
enzymes that catalyze post-translational tyrosine sulfation of proteins
Tyrosylprotein Sulfotransferases (TPSTs)
Define
Cofactor
a substance (other than the substrate) whose presence is essential for the activity of an enzyme
What does the addition of urea do to a protein?
Denatures it by forming hydrogen bonds with the proteins that disrupted the hydrogen bonds between the amino acids
What does Lane 3 and Lane 4 tell us?
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
What are the 2 distinct stages of the action of Chymotrypsin?
- Formation of acyl-enzyme intermediate
- Hydrolysis of acyl-enzyme intermediate
What do inflammation, HIV and Malaria all have in common?
They all involve chemokine receptors
What are some situations in which the Thermodynamic Hypothesis is not valid?
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
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
Expressional proteomics
What does comparing Lane 8 and Lane 4 tell us?
Shows that CD4 is not sulfated
Immunoprecipitation worked due to lane 4 band
True or False:
Like phosphorylation, sulfation is reversible
False
Phosphorylation is reversible but sulfation is not.
What is a protein domain?
A protein domain is a spatially distinct structural component that could conceivably fold and function in isolation
Why is chymotrypsin a member of the serine protease family?
Ser is the key active site residue
True or False: Sequences of low similarity can have the same 3D structure
True
Define
Reaction intermediate
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
What does the SH2 domain do?
The SH2 domain has evolved to recognise phosphotyrosines
What conclusions can be draw based in this figure?
Both tyrosines are important but tyrosine 14 is more important to chemokine response
Define
Proteome
a set of proteins produced in an organism, system or biological context
True or False:
The transition state of a compound is stabile and can be crystallised
False
Transition state is not a stable chemical species, it has no lifetime
Tyrosylprotein Sulfotransferases are selective for tyr in what kind of environment?
Acidic (aspartic and glutamic acid)
Define
Tyrosylprotein Sulfotransferases (TPSTs)
enzymes that catalyze post-translational tyrosine sulfation of proteins
Describe the role of NAD+ in redox reactions
In a redox reaction, 2 electrons and a proton are transferred to NAD+, forming NADH
What two types of ions are formed when peptides are broken for mass spec? Which ion produces peaks?
b-type ions
y-type ions (produce peaks)
Red
Definition
the first binding site in the ribosome
A site (aminoacyl)
Define
Electrophile
positively charged or neutral species having vacant orbitals that are attracted to an electron rich centre
Which features of proteins are used for 2D Gel electrophoresis?
Isoelectric focusing (pI)
Size
What type of receptors are needed for HIV to fuse with target cells?
Chemokine receptors
What are some characteristics of enzymes?
- Enzymes work very rapidly
- Activity dependent on pH
- Activity dependent on temperature
- Enzymes are substrate specific
- Many enzymes require cofactors or coenzymes
- Enzymes can be inhibited
Definition
proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures
Chaperone
Define
E site (exit)
the third and final binding site for t-RNA in the ribosome during protein synthesis
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
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
Define
Zinc finger domains
a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) in order to stabilize the fold
What are the cleavage points for pepsin?
Leu, Phe, Trp and Tyr
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.
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.
Define
Proteomics
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
What does termination consist of?
- Release of polypeptide 2. Ribosome disassembly
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.
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.
Define
G-protein-coupled receptors
a large protein family of receptors that detect molecules outside the cell and activate internal signal transduction pathways and, ultimately, cellular responses
How many transmembrane regions do chemokine receptors have?
7
Define
Duffy Antigen and Receptor for Chemokines (DARC)
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
Which amino acids does tyrosine need to be near to be sulfated?
Aspartic acid
Glutamic acid
How many nucleotides make up a codon?
3
Definition
the second binding site for tRNA in the ribosome
P site (peptidyl)
Definition
a set of proteins produced in an organism, system or biological context
Proteome
Which cells incorporate 35S-Cys/Met? Which cells incorporate 35S-SO42-?
All cells incorporate 35S-Cys/Met
35S-SO42- only incorporated if cell contains sulfate
True or False:
Enzymes are protein catalyst
False
They are biological catalysts
Most are protein but some are RNA (i.e. proteosome)
Which PTM occurs on tyrosine residues and adds negative charges to the protein?
a) Methylation
b) Ubiquitination
c) Phosphorylation
Which PTM occurs on tyrosine residues and adds negative charges to the protein?
a) Methylation
b) Ubiquitination
c) Phosphorylation
What are the cleavage points for trypsin?
Lys and Arg
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
Catalytic residues
How are some people resistant to HIV?
Mutation of the CCR5 chemokine receptor so that HIV cannot fuse with the target cell effectively
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
Zinc finger domains
Knockout of mouse orthologues of Tyrosylprotein Sulfotransferase causes what?
- Reduced body weight
- Reduced fertility
- Early death (in double knockout)
Define
Pepsin
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.
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
Wobble position
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
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
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:
- 5 M
- 5 M
- 5 M
6 M
At which of the following denaturant concentrations does the protein have the highest Gibbs free energy?
Select one:
- 5 M
- 5 M
- 5 M
6 M
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
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
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
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
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
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
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
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
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
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
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
Fig. 2 shows…
Select one:
transition state
enzyme-product complex
covalent acyl-enzyme intermediate
nucleophilic attack by Ser195
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
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
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
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
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
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
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
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
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
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
