Protein Dynamics and Disorder Practice Flashcards

A comprehensive set of 120 flashcards covering protein dynamics, intrinsically disordered proteins, thermodynamics of the free energy landscape, and experimental methods based on the lecture notes.

What term describes the well-defined 3D structure common to many proteins?

Globular

Why are protein structures not considered static?

Because the amino acids and polypeptide chain contain chemical side chains that provide flexibility, allowing them to wobble.

What is the limitation of a crystal structure regarding protein visualization?

It provides static structures of different states but does not allow for direct visualization of movement.

On what time scale do truly fast motions like bond stretches occur?

The phentosecond time scale.

On what time scale do large folding events or domain motions occur?

The millisecond time scale.

How can fast loop motions be functionally relevant in proteins?

They can be involved in gating active sites and mediating molecular interactions.

What do disordered proteins lack?

$2^{\circ}$ or $3^{\circ}$ structure.

What computational method is used to visualize the slowest, 'floppiest' motions of a protein?

Normal mode calculation

What term is used to describe the wobbly, whole-domain motion of a protein?

Breathing

Are disordered proteins equivalent to unfolded proteins?

No, they are natively disordered.

How do mutation rates differ in disordered proteins compared to ordered ones?

Disordered proteins have a faster mutation rate and can tolerate more mutations.

In what type of organisms are disordered proteins more enriched?

Eukaryotes (versus prokaryotes).

What is a key biophysical property of disordered proteins that affects ligand binding and catalysis?

A very high degree of native molecular flexibility.

What does 'FEL' stand for in the context of protein dynamics?

Free energy landscape

What determines the stability of a protein on the free energy landscape?

The free energy minimum; the lower the $\Delta G$, the more stable the protein.

Where on the free energy scale would flexible and mobile proteins typically be found compared to stable globular ones?

A little bit further up the scale, in a 'halfway house' rather than at a minimum.

What does a lack of bonds formed in a protein indicate about its position on the FEL?

The protein is further up the free energy scale and is more flexible.

What is the thermodynamic measure of disorder in a protein system?

Conformational entropy

The tendency of hydrophobic residues to exclude solvent is known as what?

The hydrophobic effect

Why is the hydrophobic effect entropically favorable for the overall system?

Because water clustering around hydrophobic residues is entropically unfavorable.

Which thermodynamic contributions favor a protein being structured and defined?

Enthalphic contributions (Hydrogen bonds and electrostatic interactions).

What does a negative enthalphic contribution indicate in terms of protein stability?

Bond forming, which favors a negative $\Delta G^{\circ}$.

How do you maximize flexibility and disorder in a protein thermodynamically?

Minimize the contribution of $\Delta H$ (less bonding) and maximize the contribution from conformational entropy $\Delta S$.

What is the standard Gibbs free energy equation provided in the transcript?

$$\Delta G^{\circ} = \Delta H - T\Delta S$$

What do each thermodynamic minima on the FEL correspond to?

A different structure of the protein.

What are transient structures on the pathway to a folded protein structure called?

Molten globule

Where are molten globules found on the Free Energy Landscape?

Around the middle of the FEL, where they are a little bit structured and a little bit unfolded.

How does a protein become an intrinsically disordered protein (IDP) according to the FEL model?

If a thermodynamic minimum is very deep (a trap) and there is not enough environmental energy to climb out.

How does the transcript define IDPs in relation to the folding pathway?

Very deep thermodynamic minima on the pathway to a much more folded protein.

What two factors define the Free Energy Landscape?

The amino acid sequence and the environment (the 'bath').

What environmental factors modulate the thermodynamics and occupied minima of the FEL?

Charges and electrostatics.

What is the phenomenon where the inside of a cell is completely packed and viscous?

Macro molecular crowding

How does macro molecular crowding affect protein structure?

It is extraordinarily stabilizing.

Why is studying molecular flexibility 'in cellulo' remarkably difficult?

Due to a lack of tools.

Does the presence of chaperones change the FEL permanently?

No, they only modulate the FEL during the period of interaction.

How do chaperones modulate the FEL during interaction?

By excluding solvent or driving hydrophobic collapse.

The propensity for structural disorder is linked to what properties of amino acids?

Physical and chemical properties.

Why are 'Hydroponic' (aromatics) residues more likely to be structured?

The hydrophobic effect drives collapse/core formation.

Why are charged and polar residues more likely to be unstructured/disordered?

They preferentially form stabilizing bonds with water, decreasing the disorder of the surrounding environment.

Which amino acid is described as the 'worst for structure'?

Proline

Why does Proline tend to break up structured elements?

Because of its rigid and planar structure.

What is the coded source of protein disorder?

The amino acid sequence.

Disordered sequences are enriched in which types of residues?

Polar and charged residues (fewer hydrophobic and aromatics).

What is the typical length of short disordered regions?

Less than 100 amino acids (<100).

How do protein-protein interactions differ between well-structured and disordered proteins?

Ordered typically use hydrophobic versus polar interactions; disordered often favor polar interactions.

Compare the number of binding partners for ordered versus disordered proteins.

Ordered have a small number of partners; disordered exhibit promiscuous binding.

How does binding affinity and specificity differ in disordered proteins?

They have high specificity but low affinity.

What happens to the structure of a disordered protein upon ligand binding?

It undergoes coupled folding and binding (loses disorder).

In which pathways are disordered proteins generally found in eukaryotes?

Signalling pathways.

Why is the low affinity of disordered protein interactions functionally important?

It allows for promiscuous and transient interactions in signalling cascades.

What does 'MoRF' stand for?

Molecular Recognition Feature

What is a MoRF?

A short structural feature (~30 AA) which folds on recognition of a ligand (disorder-to-order transition).

Where are the few hydrophobic residues in a disordered protein often located?

At the site of MoRFs.

Why are IDPs hypothesized to be enriched in eukaryotic signaling pathways compared to prokaryotes?

Due to increasing signaling complexity requiring dynamic, low-affinity interactions.

What is the 'fly-casting mechanism'?

A model suggesting disordered states have a larger capture radius, leading to faster on-rates for binding.

According to the fly-casting model, why do IDPs have greater capture radii?

Due to greater chain flexibilities.

In the fly-casting model, what happens after a flexible region weakly binds its partner?

It reels in the binding partner while completing folding simultaneously.

High conformational entropy allows IDPs to do what regarding conformational space?

Sample a very broad range of conformational space.

The fly-casting mechanism acts like what familiar object?

A fishing line

How does ligand interaction specifically affect the FEL in the fly-casting model?

It alters the FEL to drive a more folded, compacted, and stable architecture.

Name five techniques for studying structural disorder listed in the transcript.

NMR, Crystallography, Circular dichrosism, Fluorescence spectroscopy, and Single molecule detection.

Name a fine review of disordered proteins mentioned in the transcript.

Dosztányi (on moodle).

Which study is cited regarding whether proteins are disordered in the cell?

Senske M. 2014 J Am Chem Soc. 136, 9036.

What represents the illusion of dynamics in structurally disordered proteins?

Rapid traversal of small dynamic barriers between multiple thermodynamic minima.

How was the 'cute movie' of normal modes described in terms of its composition?

A set of snapshots of different conformational states and the pathways between them.

What is the primary function of the protein p53?

Transactional regulator controlling the cell cycle.

Name four proteins p53 interacts with.

S100, MDM2, Cyclin, and CDK.

Why is the whole p53 protein difficult to crystallize?

It is so flexible that high conformational entropy thermodynamically disfavours crystallogenesis.

What technique can solve structural motifs in p53 even if they are heterogeneous?

NMR (Nuclear Magnetic Resonance).

What does structural disorder allow p53 to do regarding its partners?

Interact with multiple different partners by modulating its structure (e.g., adopting helical structures).

How does p53 reduce genome size and metabolic burden?

By 'doing more with less'—one flexible regulator interacting with many different proteins.

Which terminal region of p53 contains its binding sites and is disordered?

N-terminal region

What secondary structure does the binding segment of p53 adopt when interacting with MDM2?

An well-defined $\alpha-helix$.

What type of enzyme is MDM2?

An E3 ligase

Which three specific residues in p53 mediate the interaction with MDM2?

Three hydrophobic/aromatic residues.

What are 'Date hubs' in the context of p53?

Proteins that bind to different proteins at different times.

What four different secondary structures can the same region of p53 adopt depending on the partner?

Helix, sheet, and two different irregular structures.

Describe the 14-3-3 protein strategy for interacting with multiple targets.

It maintains a fixed structure but is 'functionally flexible,' forcing partners to change conformation to fit its determinants.

Define the 'One to many' model of protein interaction.

A protein changes its own structure to fit different ligands.

Define the 'Many to one' model of protein interaction.

A protein forces different ligands to change their structure to fit its own fixed conformation.

In ordered interactions, what usually drives ligand binding?

Enthalpy (formation of H-bonds and electrostatic interactions).

What is the major thermodynamic penalty disordered proteins face during ligand binding?

A large entropic penalty because the protein must fold as it binds.

How does the $\Delta G^{\circ}$ for ligand binding in disordered proteins compare to ordered proteins?

It is smaller (less negative), meaning the binding is weaker.

What does it mean that specificity is 'uncoupled' from binding strength in IDPs?

They can be highly specific for a target despite having low affinity and transient (high off-rate) interactions.

In a simple ordered interaction, how is the kinetic scheme modeled?

As a one-step reversible process.

What determines the varying on and off rates for a disordered protein interaction?

The thermodynamic starting point (which conformational state it is in on the FEL).

What is the terminal well-structured binary complex in a disordered kinetic scheme called?

PBT

What protein's function is dependent on its mobility while shuffling electrons?

Cytochrome P450 reductase

In crystallography, what does a lack of density suggest?

Structural disorder

What does a high B-factor indicate in a crystal structure?

Mobility and dynamics

How can researchers visualize different states (open and closed) of Cytochrome P450 reductase using crystallography?

By changing crystallogenesis conditions (e.g., salt concentration) to move through different minima on the FEL.

What is the significance of domains not being connected in a crystal structure?

They are connected by flexible, loose, or disordered loops too flexible to solve.

What specific type of Circular dichroism is used to look at secondary structure?

FAR UV

What defines Circular dichroism (CD)?

The differential absorption of left and right circularly polarized light.

In a CD spectrum, what is a 'random coil' indicative of?

Disorder

What is the primary limitation of CD for assessing protein structure?

It is non-quantitative for subtle changes and only useful for extreme cases.

What does ANS stand for in fluorescence spectroscopy?

1-anilino-8-napthalene sulfonate

ANS binds most strongly to which state?

Molten globules

What happens to ANS fluorescence when it binds to unstructured content?

There is a 200-fold increase in fluorescence and a blue shift in emission.

Which residues can form ionic pairs with ANS?

Arg, Lys, and His