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Biomolecular Interactions

2025-06-18T08:07:10Z

R0905235: /* Examenvragen */

[[Categorie: Mabb]]

== Vakinformatie ==
Biomolecular Interactions

ECTS-fiche: https://onderwijsaanbod.kuleuven.be/syllabi/e/G0G77AE.htm

== Examenvragen ==
===17 june 2025===

*Prof. Mizuno
*#Describe the principles of FRET
*#Explain how you can use fluoresence lifetime to quantify FRET.
*#Give one example analyzing biomolecular interactions with FRET and explain.
*Prof. Ismail
*#Two proteins are researched to see if they interact. The Yeast-two-hybid assay is positive but when an Affinity Purification is performed there is no interaction found. Give 3 reasons why this could happen and explain what you would do to test this (controles) (9/20)
*# A student wants to research the KD of a protein that is estimated around 1 picomolar. He read that for an ITC, concentrations of 10µM are recommended. He decides that ITC is a good technique to determine the KD. Do you agree with the student? explain (2/20)
*#Two Students perform an steady state Fluorescence Anisotropy assay on two proteins to determine the KD. They use the same buffer, same concentration and same machine, yet they both have different KD values. Give two reasons why this could be the case. (4/20)
*#Explain how proteins can interact specifically with DNA. (5/20)
*Dr. Porras
*#Why is the stucture of carbohydrates important for its interactions. Give examples
*#Compare carbohydrates with Amino Acids and Proteins
*#How do proteins interact with carbohydrates. Give examples and discuss for the different types how water effects them.
*#Why is the N-glycosilation different on different cells, even in differen physiological conditions?
*#What is Bio-Orthogonal chemistry, why is it usefull to study Glycome?

===18 june 2024===
*Prof. Van den Ende: protein/carbohydrate interactions can cause many diseases:
*#Explain in great detail the exact nature of this particular type of protein/carbohydrate interactions at the molecule/atom level
*#Select two different diseases to situate the importance of protein/carbohydrate interactions. Explain the context of initiation and progress of these two diseases. What strategies can be used to prevent these protein/carbohydrate interactions and counteract disease development?
*Prof. Mizuno
*#Describe the principles of BRET
*#Summarize similarrities and differences (also advantages and drawbacks) of BRET over FRET.
*#Give one example analyzing biomolecular interactions with BRET and explain.
*Prof. Ismail
*#You're studying a newly discovered human protein in the lab and want to know what other proteins might interact with it. (10/20)
*#*Propose a method to identify potential interactions.
*#*Why did you choose this method?
*#*What are the limitations of this method?
*#*What are the experimental controls you need to do whule executing this method?
*#*Can you propose (only the name) another method that can overcome at least one of the limitations you mentioned before?
*#The figure below depicts 3 simulated curves of 3 isothermal titration calorimetry (ITC) experiments to determine the values: binding affinity Kd and stoichiometry (n). Each curve represents an experiment where the concentration of protein in the cell is different as indicated by the c-value on each curve. Indicate on each graph which values can be estimated with confidence. (the picture was the same as on the slides about the c-value effect) (2/20)
*#Mention 4 applications of Y2H system (2/20)
*#A student has 2 proteins, one of 4kDa and one of 50 kDa and wanted to determine the Kd. A colleague advised them to do a fluorescently label the 50 kDa protein and anisotropy assy. The student didn't find this a good idea. Do you agree with the student? (2/20)
*#Facilitated diffusion of proteins on DNA decreases the search time of proteins for their target sites, discuss. (4/20)

=== 21 juni 2022 ===
#Van den Ende: Protein-carbohydrate interactions can cause many diseases: what is the nature of these interactions? Give two examples of diseases with protein-carbohydrate interactions and say how you could stop their progress.
#Mizuno: Explain the principle of BRET. What are the advantages, disadvantages, similarities and differences with FRET? Give an example of the study of biomolecular interactions using BRET.
#Ismail: Explain the phenomenon that proteins find their target so quickly (quicker than observed in 3D diffusion).
#Robben: You want to study the interactome between virus and host (both genomes are known). Which strategy would you apply? How would you adapt the method to detect weak interactions?

=== 17 juni 2020 (Corona exam: 3 hours)===
#Van den Ende: protein-carbohydrate interactions can cause many diseases? What is the nature of these interactions? Give two examples of diseases with protein-carbohydrate interactions and say how you could stop their progress.
#Mizuno: Given the equations describing kT, explain three factors affecting FRET efficiency and how (some of them) have to be taken into account when designing molecular sensors. Give two examples about molecular sensors using FRET, one with intermolecular and other with intramolecular FRET.
#Robben: you need to produce mouse monoclonal antibodies against the SARS-CoV-2. The spike protein is provided. Describe how would you proceed using phage display. What experimental measures would you take to ensure maximal affinity of the antibodies?

=== 14 juni 2019===
#Van den Ende: protein-carbohydrate interactions can cause many diseases? What is the nature of these interactions? Give examples of diseases with protein-carbohydrate interactions.
#Mizuno: Describe FRET and fluoresence lifetime, give examples to explain these concepts.
#Robben: Why doesn't the classical yeast two hybrid system work when one of the proteins is a membrane protein? Which alternative systems do you know would work? Give major advantages or disadvantages.
(Oral with Wim)

=== 15 juni 2018===

#Robben: Wat zijn de verschillende manieren waarop EcoRV bindt met DNA, welke methodologieën om dit te onderzoeken?
#Mizuno: Je collega ziet bij confocale microscopie dat membraaneiwit A en membraaneiwit B op de zelfde positie zichtbaar zijn en besluit dat ze dus interageren. Wat vind jij hiervan en waarom? Welke andere technieken zou je voorstellen en geef de achtergrond van deze technieken.
#Van den Ende: polypeptide-polysaccharideinteracties voor ziektes: Geef belang aan met behulp van een voorbeeld, en geef een voorbeeld van hoe dit de basis zou kunnen zijn van een therapie.

=== 26 juni 2017 ===
# Mizuno-senpai:
## Explain the principle of the Scatchard plot using the degree of association (fi)
##The two given plots depict a wild type (wt) and a mutant (cc) EGFR’s scatchard plot, with the twist that L(bound) is used instead of fi.
###Explain, how to calculate the association constant! Derive the linear, to get -K
###Give the association constant for CC! devide y intercept with x intercept, dont forget the unit! (K~0.9/12[1/pM])
###What does the x intercept mean? We can read out E0, because fi=1 at the x intercept. fi=L(bound)/E0=1 → L(bound)=E0
###Discuss the differences between WT & CC. WT curves up, which indicates an additional binding site. As for that binding site, the association constant is greater (see graph) it has a higher affinity to the substrate. This binding site is lost in the mutation. (Also this is why it is conceptually incorrect to calculate K from the y intercept on the scatchard plot)
#G. Maglia: Describe how DNA binding proteins recognize specific features in ssDNA and dsDNA.
#J. Robben: You want to develop a new ELISA assay for Zika virus. The coat protein (the antigen) is given. You decide to use phage display
##Describe how you would proceed in the lab! Procedure for antibody display (use nanobodies), biopanning, amplification etc.
##Which experimental measures would you use to ensure maximal affinity? According to those, who had the oral with him, the answer was monovalent display (so type33 or type3+3 display)
#J. Hofkens:A new theory is proposed for the structure of biological membranes, according to which it contains:
##Rafts
##Compartments that are due to the partition of the actin filaments (cytoskeleton) in the membrane
##Membrane proteins, that are present in oligomer form, or as transient dimers or bigger complexes. Describe how would you confirm this theory, with the microscopy techniques learned in class.

=== 20/06/2016 ===
#Prof. Hideaki Mizuno Sensei : Single step reversible association and fast binding followed by a slower conformational change.
#Prof. Robben : Describe by drawing diagram of genetic component of enzyme complementation system. What are the strengths and weaknesses of this system compare to the traditional transcription factor based system.
#Prof. De Maeyer : Discuss pharmacophore modelling. Focus on module 2.
#Prof.Hofkens : Imaging of receptor molecules in neural ring synapse in C.elegans.

=== 20/08/2016 ===
# Prof. Hideaki Mizuno Sensei : Define the Binding equation. Derive the partition function and the association constant. Provide the Hill Plot and Scatchard plot of a single binding site. Explain what kind of information can be obtained from these plots. Explain the above analysis in the context of oxygen binding.
# Prof. Robben : Give a schematic representation of Tandem Affinity purification. Explain why sometimes cross linking is used. How is the outcome of this experiment different from 2 hybrid systems..
# Prof. De Maeyer : Discuss pharmacophore modelling. Focus on module 2(ligand receptor complex).
#Prof.Hofkens : To distinguish the type of movement of two different motor proteins

=== 21/08/2015 ===
#Prof. Hideaki Mizuno Sensei : Single step and two step binding kinetics models. Basically all the equations. Know about the rate constants, what do they mean and how do you obtain them. Does not give any equation or formula.
#Prof. Giovanni Maglia : Explain how do proteins bind to a specific sequence in the major groove of the DNA.
#Prof. Marc De Maeyer: Discuss pharmacophore modelling. Focus on module 3.
# Prof. Johan Robben :· Classic yeast two-hybrid models are not viable if one or both proteins involved are cellular membrane proteins. Explain. · Give an example schematically on how would you model it correctly.

Biomolecular Interactions

2025-06-17T13:26:46Z

R0905235: /* Examenvragen */

[[Categorie: Mabb]]

== Vakinformatie ==
Biomolecular Interactions

ECTS-fiche: https://onderwijsaanbod.kuleuven.be/syllabi/e/G0G77AE.htm

== Examenvragen ==
===17 june 2025===

*Prof. Mizuno
*#Describe the principles of FRET
*#Explain how you can use fluoresence lifetime to quantify FRET.
*#Give one example analyzing biomolecular interactions with FRET and explain.
*Prof. Ismail
*#2 proteins are researched to see if they interact. The Yeast-two-hybid assay is positive but when an Affinity Purification is performed there is no interaction found. Give 3 reasons why this could happen and explain what you would do to test this (controles) (9/20)
*# A student wants to research the KD of a protein that is estimated around 1 picomolar. He read that for an ITC, concentrations of 10µM are recommended. He decides that ITC is a good technique to determine the KD (do you agree with the student? explain (2/20)
*#Two Students perform an steady state Fluorescence Anisotropy assay on two proteins to determine the KD. They use the same buffer, same concentration and same machine, yet they both have different KD values. Give two reasons why this could be the case. (4/20)
*#Explain how proteins can interact specifically with DNA. (5/20)
*Prof Porras
*#Why is the stucture of carbohydrates important for its interactions. Give examples
*#Compare carbohydrates with Amino Acids and Proteins
*#How do proteins interact with carbohydrates. Give examples and discuss for the different types how water effects them.
*#Why is the N-glycosilation different on different cells, even in differen physiological conditions?
*#What is Bio-Orthogonal chemistry, why is it usefull to study Glycome?

===18 june 2024===
*Prof. Van den Ende: protein/carbohydrate interactions can cause many diseases:
*#Explain in great detail the exact nature of this particular type of protein/carbohydrate interactions at the molecule/atom level
*#Select two different diseases to situate the importance of protein/carbohydrate interactions. Explain the context of initiation and progress of these two diseases. What strategies can be used to prevent these protein/carbohydrate interactions and counteract disease development?
*Prof. Mizuno
*#Describe the principles of BRET
*#Summarize similarrities and differences (also advantages and drawbacks) of BRET over FRET.
*#Give one example analyzing biomolecular interactions with BRET and explain.
*Prof. Ismail
*#You're studying a newly discovered human protein in the lab and want to know what other proteins might interact with it. (10/20)
*#*Propose a method to identify potential interactions.
*#*Why did you choose this method?
*#*What are the limitations of this method?
*#*What are the experimental controls you need to do whule executing this method?
*#*Can you propose (only the name) another method that can overcome at least one of the limitations you mentioned before?
*#The figure below depicts 3 simulated curves of 3 isothermal titration calorimetry (ITC) experiments to determine the values: binding affinity Kd and stoichiometry (n). Each curve represents an experiment where the concentration of protein in the cell is different as indicated by the c-value on each curve. Indicate on each graph which values can be estimated with confidence. (the picture was the same as on the slides about the c-value effect) (2/20)
*#Mention 4 applications of Y2H system (2/20)
*#A student has 2 proteins, one of 4kDa and one of 50 kDa and wanted to determine the Kd. A colleague advised them to do a fluorescently label the 50 kDa protein and anisotropy assy. The student didn't find this a good idea. Do you agree with the student? (2/20)
*#Facilitated diffusion of proteins on DNA decreases the search time of proteins for their target sites, discuss. (4/20)

=== 21 juni 2022 ===
#Van den Ende: Protein-carbohydrate interactions can cause many diseases: what is the nature of these interactions? Give two examples of diseases with protein-carbohydrate interactions and say how you could stop their progress.
#Mizuno: Explain the principle of BRET. What are the advantages, disadvantages, similarities and differences with FRET? Give an example of the study of biomolecular interactions using BRET.
#Ismail: Explain the phenomenon that proteins find their target so quickly (quicker than observed in 3D diffusion).
#Robben: You want to study the interactome between virus and host (both genomes are known). Which strategy would you apply? How would you adapt the method to detect weak interactions?

=== 17 juni 2020 (Corona exam: 3 hours)===
#Van den Ende: protein-carbohydrate interactions can cause many diseases? What is the nature of these interactions? Give two examples of diseases with protein-carbohydrate interactions and say how you could stop their progress.
#Mizuno: Given the equations describing kT, explain three factors affecting FRET efficiency and how (some of them) have to be taken into account when designing molecular sensors. Give two examples about molecular sensors using FRET, one with intermolecular and other with intramolecular FRET.
#Robben: you need to produce mouse monoclonal antibodies against the SARS-CoV-2. The spike protein is provided. Describe how would you proceed using phage display. What experimental measures would you take to ensure maximal affinity of the antibodies?

=== 14 juni 2019===
#Van den Ende: protein-carbohydrate interactions can cause many diseases? What is the nature of these interactions? Give examples of diseases with protein-carbohydrate interactions.
#Mizuno: Describe FRET and fluoresence lifetime, give examples to explain these concepts.
#Robben: Why doesn't the classical yeast two hybrid system work when one of the proteins is a membrane protein? Which alternative systems do you know would work? Give major advantages or disadvantages.
(Oral with Wim)

=== 15 juni 2018===

#Robben: Wat zijn de verschillende manieren waarop EcoRV bindt met DNA, welke methodologieën om dit te onderzoeken?
#Mizuno: Je collega ziet bij confocale microscopie dat membraaneiwit A en membraaneiwit B op de zelfde positie zichtbaar zijn en besluit dat ze dus interageren. Wat vind jij hiervan en waarom? Welke andere technieken zou je voorstellen en geef de achtergrond van deze technieken.
#Van den Ende: polypeptide-polysaccharideinteracties voor ziektes: Geef belang aan met behulp van een voorbeeld, en geef een voorbeeld van hoe dit de basis zou kunnen zijn van een therapie.

=== 26 juni 2017 ===
# Mizuno-senpai:
## Explain the principle of the Scatchard plot using the degree of association (fi)
##The two given plots depict a wild type (wt) and a mutant (cc) EGFR’s scatchard plot, with the twist that L(bound) is used instead of fi.
###Explain, how to calculate the association constant! Derive the linear, to get -K
###Give the association constant for CC! devide y intercept with x intercept, dont forget the unit! (K~0.9/12[1/pM])
###What does the x intercept mean? We can read out E0, because fi=1 at the x intercept. fi=L(bound)/E0=1 → L(bound)=E0
###Discuss the differences between WT & CC. WT curves up, which indicates an additional binding site. As for that binding site, the association constant is greater (see graph) it has a higher affinity to the substrate. This binding site is lost in the mutation. (Also this is why it is conceptually incorrect to calculate K from the y intercept on the scatchard plot)
#G. Maglia: Describe how DNA binding proteins recognize specific features in ssDNA and dsDNA.
#J. Robben: You want to develop a new ELISA assay for Zika virus. The coat protein (the antigen) is given. You decide to use phage display
##Describe how you would proceed in the lab! Procedure for antibody display (use nanobodies), biopanning, amplification etc.
##Which experimental measures would you use to ensure maximal affinity? According to those, who had the oral with him, the answer was monovalent display (so type33 or type3+3 display)
#J. Hofkens:A new theory is proposed for the structure of biological membranes, according to which it contains:
##Rafts
##Compartments that are due to the partition of the actin filaments (cytoskeleton) in the membrane
##Membrane proteins, that are present in oligomer form, or as transient dimers or bigger complexes. Describe how would you confirm this theory, with the microscopy techniques learned in class.

=== 20/06/2016 ===
#Prof. Hideaki Mizuno Sensei : Single step reversible association and fast binding followed by a slower conformational change.
#Prof. Robben : Describe by drawing diagram of genetic component of enzyme complementation system. What are the strengths and weaknesses of this system compare to the traditional transcription factor based system.
#Prof. De Maeyer : Discuss pharmacophore modelling. Focus on module 2.
#Prof.Hofkens : Imaging of receptor molecules in neural ring synapse in C.elegans.

=== 20/08/2016 ===
# Prof. Hideaki Mizuno Sensei : Define the Binding equation. Derive the partition function and the association constant. Provide the Hill Plot and Scatchard plot of a single binding site. Explain what kind of information can be obtained from these plots. Explain the above analysis in the context of oxygen binding.
# Prof. Robben : Give a schematic representation of Tandem Affinity purification. Explain why sometimes cross linking is used. How is the outcome of this experiment different from 2 hybrid systems..
# Prof. De Maeyer : Discuss pharmacophore modelling. Focus on module 2(ligand receptor complex).
#Prof.Hofkens : To distinguish the type of movement of two different motor proteins

=== 21/08/2015 ===
#Prof. Hideaki Mizuno Sensei : Single step and two step binding kinetics models. Basically all the equations. Know about the rate constants, what do they mean and how do you obtain them. Does not give any equation or formula.
#Prof. Giovanni Maglia : Explain how do proteins bind to a specific sequence in the major groove of the DNA.
#Prof. Marc De Maeyer: Discuss pharmacophore modelling. Focus on module 3.
# Prof. Johan Robben :· Classic yeast two-hybrid models are not viable if one or both proteins involved are cellular membrane proteins. Explain. · Give an example schematically on how would you model it correctly.