Chris' TBL Questions Flashcards
(37 cards)
What methods can be used to detect proteins/enzymes at the single molecule level?
a) Steady-state kinetics
b) Total internal reflectance fluorescence microscopy
c) Atomic force microscopy
d) X-ray crystallography
e) molecular dynamics simulations
b) Total internal reflectance fluorescence microscopy
c) Atomic force microscopy
d) X-ray crystallography
e) molecular dynamics simulations
How can we immobilise proteins for singular molecule measurements?
a) Streptavidin tags
b) Histidine tags
c) Membrane mediated
d) Thiol linkage
e) All of the above
e) All of the above
What is a primary function of protein flexibility in biological systems?
a) Maintaining a rigid, unchanging structure
b) Enabling conformational changes for enzyme catalysis, protein-protein interactions and allosteric enzyme activation
c) Preventing protein degradation
d) Facilitating DNA replication
b) Enabling conformational changes for enzyme catalysis, protein-protein interactions and allosteric enzyme activation
What is the purpose of using Forster resonance energy transfer (FRET) in a single molecule studies of protein folding?
a) To enhance the fluorescence signal of the protein
b) To measure distances and conformational changes within a protein
c) To study the interaction between an enzyme and its substrate
d) To observe the real-time movement of proteins inside a cell
a) To enhance the fluorescence signal of the protein
b) To measure distances and conformational changes within a protein
What is a potential drawback of non-specific enzyme absorption onto a surface?
a) It can lead to increased enzyme activity
b) It can cause the enzyme to orient with the active site toward the surface, preventing substrate binding
c) It always results in more accurate measurements
d) It makes it easier to study single-molecule behaviour
b) It can cause the enzyme to orient with the active site toward the surface, preventing substrate binding
What is a key advantage of using the cofactor itself as a fluorescent reporter in enzyme studies?
a) It increases the signal-to-noise ratio
b) It allows for the determination of the durations of individual states along the turnover cycle
c) It eliminates the need for extrinsic fluorescent labels
d) It provides higher spatial resolution
e) It allows us to detect the intrinsic rates of chemical turnover, free of complexity from other steps
c) It eliminates the need for extrinsic fluorescent labels
e) It allows us to detect the intrinsic rates of chemical turnover, free of complexity from other steps
In a single-molecule fluorescence detection, what is a primary challenge to extracting accurate data?
a) The low number of photons emitted by a single molecule
b) The high sensitivity of detectors to background light
c) The fact that many molecules can fluoresce or scatter light.
d) The need for specialised fluorescent dyes
e) All of the above
e) All of the above
A researcher is investigating the catalytic activity of a single enzyme using single-molecule fluorescence microscopy. They observe that the enzyme’s turnover rate fluctuates over time. They want to determine if these fluctuations are due to variations in the enzyme’s conformation or due to stochastic binding and unbinding of an inhibitor. Based on the provided sources, which experimental approach and subsequent analysis would be most suitable to investigate the following questions?
- To determine if the fluctuations in activity are due to conformational changes in the enzyme
- To measure the kinetic parameters of the enzyme in the presence and absence of a suspected inhibitor
- To test whether the inhibitor acts in a competitive or uncompetitive manner
a) Using single-molecular force spectroscopy (SMFA) with an Atomic Force Microscope (AFM) to generate force-extension curves (FECs) that reveal the mechanical unfolding and refolding of the enzyme, using this data to map the energy landscape of the enzyme in the presence and absence of inhibitor at a single temperature
b) Employing a Total Internal Reflection Fluorescence (TIRF) microscope with a fluorogenic substrate, analysing the time-intensity trajectories to extract dwell times for substrate turnover events, fitting the data to the exponential functions to determine the catalytic rate constants in the presence and absence of the inhibitor, and plotting the inverse turnover rate against inhibitor concentration to determine mode of inhibition.
c) Utilising a Whispering Gallery Mode (WGM) sensor with gold nanorods to which the enzyme is attached, analysing the time traces of the WGM resonance wavelength to extract arrival times between catalytic events, performing measurements at different inhibitor concentrations to determine binding and unbinding rates, and using a stochastic model of enzyme inhibition to determine the mechanism of inhibition
d) Performing ensemble kinetic assays in a spectrophotometer to measure the average product formation rate in the presence and absence of the inhibitor at different substrate concentrations, using Lineweaver-Burk plots to determine the Michaelis-Menten parameters and the mode of inhibition at a single temperature.
A researcher iteam is investigating the refolding of a membrane protein, the bacteriorhodopsin (bR) using single-molecule force spectroscopy (SMFS). They are particularly interested in understanding the protein’s energy landscape and the role of specific interactions in stabilising its native state. They are using an AFM tip to apply force to the protein and have made the following observations.
- During the initial unfolding of bR, they observe multiple intermediate states, including some involving the unfolding of only a few amino acids
- They find that the unfolding pathways and the stability of intermediates can vary depending on the direction of the applied force and the specific attachment point of the AFM tip to the protein
- They also observe that the presence of retinal stabilises a previously undetected intermediate during unfolding
Based on these observations, which of the following approaches would be most effective for the researchers to gain a comprehensive understanding of the energy landscape of bR and the role of specific interactions
a) Using a non-specific tip attachment to the protein and analysing only the unfolding force curves
b) Using a specific attachment chemistry to link the AFM tip to a particular residue, combined with both unfolding and refolding measurements
c) Using low force to unfold the protein and only measuring the transitions between native and unfolded states
d) Studying the protein in a detergent micelle and analysing only the unfolding face curves
b) Using a specific attachment chemistry to link the AFM tip to a particular residue, combined with both unfolding and refolding measurements
Which experimental method provides an important insight into flexibility from sampling of a number of structures and refers to B-factors as flexibility reporters?
a) Limited proteolysis
b) Mass spectrometry
c) X-ray crystallography
d) Molecular simulations
c) X-ray crystallography
An electron is more likely to transfer from a donor to an acceptor when they are…
a) Far apart and energetically degenerate
b) Close together and energetically different
c) Where the wavefunction is greater
d) If the momentum of the electron is very large
c) Where the wavefunction is greater
What is a primary function of protein flexibility in biological systems?
a) Maintaining a rigid, unchanging structure
b) enabling conformational changes for enzyme catalysis, protein-protein interactions and allosteric activation
c) Preventing protein degradation
d) Facilitating DNA replication
b) enabling conformational changes for enzyme catalysis, protein-protein interactions and allosteric activation
Do we want a rigid or a fluctuating active site structure?
Yes, sometimes
The heat capacity of catalysis arises from…
a) The decay of vibrational modes as the transition state converts to the product
b) Multiple catalytically competent reactive configurations
c) A difference in the distribution of vibrational modes between the ground state and transition state
d) A quantum mechanical effect that arises during tunnelling reactions
c) A difference in the distribution of vibrational modes between the ground state and transition state
What is a good example of issues with intrinsically disordered proteins?
Large protein aggregates that give rise to Alzheimer’s are caused by hydrogen binding in proteins that have extremely flexible structures.
Structurally disordered proteins are prone to misfolding - diseased state
Under what conditions does transition state theory break down?
a) Assessing only catalytic properties of enzymes
b) Sequence diversification without assessing dynamics
c) Applying knowledge of dynamic properties to guide sequence modification
d) Ignoring the dynamic properties of variants or homologs
c) Applying knowledge of dynamic properties to guide sequence modification
What is the goal of enhanced sampling methods in molecular dynamics simulations?
a) To reduce the computational cost of simulations
b) To accelerate the sampling of the protein configurations
c) To eliminate the need for reaction coordinates
d) To study protein structures only in their crystal form
b) To accelerate the sampling of the protein configurations
Can’t run models that last for the time necessary to access all conformations, this aims to try and fix that.
What is the effect of macro-molecular crowding?
a) It decreases enzyme activity
b) It destabilises closed conformations
c) It stabilizes closed conformations through steric confinement
d) It has no significant effect on protein functions
c) It stabilizes closed conformations through steric confinement
You are studying an enzyme, MaIL, and want to engineer it to function more efficiently at higher pressures. You have two variants: the wild-type and a mutant. You perform experiments and simulations, gathering data on their dynamics and catalytic activity under varying conditions.
Which observation(s) below would suggest that the WT enzyme is more flexible than the mutant and that this flexibility is functionally relevant?
a) The enzyme activity increases with temperature
b) The enzyme activity varies with pressure
c) The X-ray crystal structure of the wild-type have a higher B-factor than the mutant
d) Molecular dynamics simulations show that the wild-type has a bigger variance in the RMSF compared to the mutant
b) The enzyme activity varies with pressure
You are trying to engineer an enzyme to be more stable at high temperatures for industrial biocatalysis. You know that protein dynamics play a role in stability, and you want to use computational methods to guide your design.
Which strategy would be mots effective in identifying mutations that increase the enzyme’s thermostability while maintaining its activity?
a) Focussing on the active site, introducing mutations that increase rigidity based on the crystal structure
b) Using molecular dynamics (MD simulations to identify flexible regions, then introducing mutations to increase compactness in these regions, while ensuring active-site flexibility is maintained.
c) Performing ancestral sequence reconstruction (ASR) to identify ancestral enzymes with higher thermostability and then introducing those ancestral residues into the modern enzyme’s active site.
d) Employing a machine learning (ML) algorithm trained on protein sequence and structure data to predict stabilising mutations
e) All of the above
You are working with an enzyme and want to improve its catalytic efficiency through dynamic engineering. You generate several variants of the enzyme and characterise their kinetic structural and dynamic properties.
Following the characterisation of your enzyme variants, what is the next step in a dynamic engineering workflow?
a) Discarding all variants that do not show improved activity.
b) Implementing machine learning algorithms to predict the best mutations
c) Linking dynamic information to catalytic function in the variants to rationalise the relationship between sequence modification, dynamics and enzyme activity
d) Focussing on improving the enzyme’s structural stability
c) Linking dynamic information to catalytic function in the variants to rationalise the relationship between sequence modification, dynamics and enzyme activity
How often are plots of enzyme temperature dependence curved?
a) Never
b) Always
c) Sometimes
b) Always
If we accept that heat capacity theory is true. This value will almost never be truly 0.
Which condition causes the Eyring equation to poorly describe enzyme catalysis?
a) Temperature around 100 C
b) The mechanism is QM tunnelling
c) Multiple reactive configurations of the active site
d) A planar transition state
b) The mechanism is QM tunnelling
No longer described by thermodynamics
c not correct because each configuration follows its own Eyring equation
What are n-state models for enzymes?
a) Enzyme mechanisms with multiple substrates
b) A description of single molecule reactivity
c) The presence of multiple catalytically active sites
d) A range of chemical mechanisms
c) The presence of multiple catalytically active sites
