ATP and GAP Binding Affinity Quiz

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Questions and Answers

What is the value of KD for ATP based on the given data?

0.5 mM

7.7 µM (correct)

15 mM

152 µM

Which compound has a higher KD value, ATP or GAP?

Both have the same KD

GAP (correct)

ATP

Neither has a measurable KD

What is the numerical value of KD for GAP?

8 µM

5 mM

10 µM

152 ± 19 µM (correct)

How does the n value relate to the binding affinity of ATP?

It suggests that binding is cooperative with n > 1 Signup and view all the answers

What does a lower KD value indicate regarding the affinity of a ligand for its receptor?

Higher affinity Signup and view all the answers

What is the effect of increasing NaCl concentrations on ATP-binding affinity in OEP21?

A 120-fold drop in binding affinity Signup and view all the answers

What role does Mg2+ play in the context of ATP interactions within the cell?

Decreases the negative charge density of ATP Signup and view all the answers

How does the deletion of L5 affect the high-affinity binding site in OEP21?

Has no effect on the high-affinity binding site Signup and view all the answers

What was the observed effect of GAP on NMR signals in the presence of OEP21?

Line broadening of NMR signals Signup and view all the answers

What is the translocation frequency of GAP with OEP21ΔL5 compared to with OEP21?

About ten times higher than with OEP21 Signup and view all the answers

What is the consequence of GAP binding, as indicated by the NMR analysis?

Slows down translocation Signup and view all the answers

Which positively charged residues are provided by L5 that interact with OEP21?

Lys76 and Arg79 Signup and view all the answers

What is the effect of applying a 180 mV membrane potential during the simulation?

It facilitates translocation of GAP Signup and view all the answers

What effect does the addition of L5 have on translocation?

It increases translocation. Signup and view all the answers

What is the effect of Mg2+ on translocation?

It decreases translocation. Signup and view all the answers

In which part of the cell is Loop 5 located?

Cytosol Signup and view all the answers

What molecular weight is indicated for the sample showing no translocation?

1,688 Da Signup and view all the answers

Which of the following amino acids is indicated near Loop 5?

Lysine Signup and view all the answers

What is the primary focus of the research article mentioned?

Mechanisms of ion translocation in cells. Signup and view all the answers

What does the acronym GAP refer to in the context given?

GTPase Activating Protein Signup and view all the answers

What were the two controls measured alongside the translocation assays?

Time and concentration Signup and view all the answers

Which amino acid is found at position E149?

Glutamic acid Signup and view all the answers

What does the presence of no translocation indicate about the experimental results?

The experimental conditions were ineffective. Signup and view all the answers

What effect do GAP and Mg2+ have on the dissociation of ATP from OEP21?

They synergistically facilitate ATP dissociation from OEP21. Signup and view all the answers

What was the IC50 value for the OEP21-ATP complex when inorganic phosphate was added?

~10 mM Signup and view all the answers

Which factor contributed to the relatively high GAP concentrations required for ATP dissociation?

Partial unspecific binding to the detergent micelle. Signup and view all the answers

How does the binding affinity of Mg2+ compare with that of ATP and phosphate?

Mg2+ interacts weakly with GAP and phosphate. Signup and view all the answers

What is indicated by the KD values for ATP determined with the set of OEP21 variants?

They show a similar high-affinity binding as the wild-type. Signup and view all the answers

What role do positive charges play in the binding of phosphorylated metabolites to OEP21?

They compensate for the removal of individual positive charges in OEP21. Signup and view all the answers

What was observed about the structural integrity of the OEP21 variants produced?

They were properly folded and showed comparable stability. Signup and view all the answers

Which variant of OEP21 was noted to have a lower binding affinity during the experiments?

ΔL5 variant Signup and view all the answers

What is the significance of a 1:1 molar ratio in the context of this data?

It indicates maximum binding efficiency. Signup and view all the answers

What does 'fold increase in KD for ATP' suggest about ATP's interaction?

A lower KD indicates a stronger binding affinity. Signup and view all the answers

Why might excess ATP affect KD values?

It leads to decreased competition and increased binding. Signup and view all the answers

What could be inferred if the fold increase in KD is significantly greater than 1?

There is likely a competition for binding sites. Signup and view all the answers

The graphical representation appears to show relationships between which variables?

Molar ratios of ATP and other ligands. Signup and view all the answers

What conclusion can be made from a lower fold increase in KD?

There is a stronger binding affinity。 Signup and view all the answers

When assessing ligand-receptor interactions, what does a KD value represent?

The concentration at which ligand binding occurs. Signup and view all the answers

How might ATP's concentration affect receptor activation?

Increased ATP can either enhance or inhibit activity. Signup and view all the answers

How does temperature likely influence the folding and activity of proteins in this context?

Lower temperatures generally enhance stability. Signup and view all the answers

Which factor could potentially disrupt KD values in ligand-receptor studies?

Increased ionic strength of the solution. Signup and view all the answers

What role does the term 'relative' play in the studied interactions?

Denotes values compared to a baseline. Signup and view all the answers

If the dataset includes terms like R1 A, what could these represent?

Labels for different receptor isoforms or variants. Signup and view all the answers

What is the implication of the term 'excess ATP' in the study?

Potential saturation of receptor sites. Signup and view all the answers

What effect does the addition of ATP have on the NMR spectral signals?

It improves the spectral signals. Signup and view all the answers

What was observed concerning the intrinsic flexibility of the protein when ATP was added?

Random coil peaks disappeared. Signup and view all the answers

Which observation indicates the stabilization effect of ATP on OEP21?

Disappearance of random coil peaks. Signup and view all the answers

What does the KD for ATP represent?

The dissociation constant for ATP binding. Signup and view all the answers

In the context of OEP21, what is the significance of the term 'solvent accessibility'?

It reflects the flexibility of specific regions of the protein. Signup and view all the answers

What might the structural indication of 'flexible L5' imply about the protein's functionality?

It is adaptable and may perform multiple roles. Signup and view all the answers

Why is trypsin mentioned in the context of this study?

It is used to cleave the protein for analysis. Signup and view all the answers

What does the presence of higher oligomer states indicate about OEP21?

It can form multiple stable complexes. Signup and view all the answers

How does the term 'unfolded' relate to the protein structure discussed?

It suggests partial loss of structural integrity. Signup and view all the answers

Which factor could potentially lower the KD for ATP binding to OEP21?

Higher concentration of ATP. Signup and view all the answers

What could be inferred about the ATP binding mechanism to OEP21?

It is likely dependent on the conformation of the protein. Signup and view all the answers

What role do molecular weight (MW) markers play in the experiments discussed?

They provide a reference for protein sizes and interactions. Signup and view all the answers

What does the acronym 'NOE' in NMR stand for?

Nuclear Overhauser Effect. Signup and view all the answers

What impact does the solvent LDAO have on the study of OEP21?

It enhances the soluble state of proteins. Signup and view all the answers

Study Notes

Structural Basis of Metabolite Transport by the Chloroplast Outer Envelope Channel OEP21

Triose phosphates (TPs) are the primary products of photosynthesis.
TPs need to be exported from chloroplasts into the cytosol, crossing both the inner and outer envelopes (IE and OE).
OEP21, a transporter protein in the chloroplast outer envelope, is the main exit pore for TPs in C3 plants.
OEP21 has a cone-shaped beta-barrel structure with a highly positively charged interior.
This positive charge allows binding and translocation of negatively charged metabolites like TPs, and to some extent, molecules up to ~1 kDa.
ATP stabilizes the OEP21 channel, keeping it open.
OEP21 has broad substrate selectivity, enabling transport of various metabolites.
The abundance of OEP21 can change in different plant species reflecting various metabolite fluxes.
OEP21 likely operates passively using a concentration gradient.
Its interaction with ATP can serve as a regulatory mechanism, potentially impacting the opening status of OEP21 channel, affecting downstream metabolic processes.

Orientation and Oligomeric State of OEP21

OEP21's funnel shape suggests an outward-rectifying orientation, with the wider opening towards the intermembrane space (IMS).
Limited proteolysis experiments with isolated chloroplast outer envelope vesicles confirmed the cytoplasmic orientation of loop 5 (L5) in OEP21. The N and C termini were located in the IMS.
OEP21 forms oligomers, with the oligomeric state potentially regulated by ATP, either impacting the degree of oligomerization or interacting dynamically with the pore.
OEP21 can form dimers and higher-order oligomers; ATP binding may lead to partial reduction in oligomer formation.
The presence of loop 5 (L5) impacts transport efficiency, with its removal leading to improved translocation.

OEP21 Metabolite Binding

OEP21's interactions with metabolites are charge-dependent, with ATP binding with a high affinity, and exhibiting a 1:1 molar ratio of interaction.
Metabolites like GAP or phosphate can compete with ATP for binding.
The presence of Mg²⁺ can modulate the binding affinity and transport kinetics of certain metabolites.

Size-Selective Metabolite Translocation

OEP21's structure permits the passage of metabolites up to ~1 kDa.
Molecules larger than ~1 kDa (vitamin B12, lysozyme) cannot transit.
The OEP21 channel's selectivity permits preferential movement of negative charges.
MD simulations suggest that GAP translocation across the OEP21 channel involves transient interactions with positively charged pore regions and translocation occurs in the microseconds-to-milliseconds timescale..
ATP can enhance or maintain the open state and thus regulate translocation of metabolites like GAP, across the pore.

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Description

Test your knowledge on the binding affinities and interactions of ATP and GAP in the context of OEP21. This quiz covers key concepts related to KD values, the impact of NaCl and magnesium ions, and the structural implications of the L5 deletion. Dive into the biochemistry behind these important cellular processes!