bioc protein nucleic acid lec 5

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Questions and Answers

What drives the rapid formation of secondary structure elements during protein folding?

The random arrangement of amino acids

Hydrophobic interactions within the protein sequence (correct)

Electrostatic repulsion between distant amino acids

Covalent bonding between side chains

How long does it typically take for very small proteins to fold?

Around 10 μs

About 100 ms

Approximately 1 μs (correct)

Less than 1 ns

Which of the following statements about the early stages of protein folding is true?

A hydrophobic core collapses rapidly (correct)

Amino acids in contact are the same as in the native structure

Secondary structure resembles its native form perfectly

The topology is often correct from the start

What role do disulfide bonds or metal ion binding play in protein folding?

They help stabilize the protein's final conformation Signup and view all the answers

What is the characteristic of the secondary structure elements formed quickly during folding?

They exhibit dynamic behaviors like extending and shrinking Signup and view all the answers

What defines a protein as 'folded'?

It is in a compact, stable, functional conformation. Signup and view all the answers

Which microstates of trypsin are the most populated?

Red is the low affinity state and green is the high affinity state. Signup and view all the answers

What role do chemical denaturants play in studying protein folding?

They destabilize the protein structure by competing for interactions. Signup and view all the answers

What happens to an unfolded protein?

It takes on many conformations, mostly unlike the native structure. Signup and view all the answers

What is the significance of slow interconversion between specific states in trypsin dynamics?

It dominates the activation/inactivation dynamics of trypsin. Signup and view all the answers

What is a characteristic of intrinsically disordered proteins (IUPs)?

They can remain in an unfolded state indefinitely. Signup and view all the answers

Which of the following roles do intrinsically unstructured proteins not typically fulfill?

Enzymatic activity with high specificity Signup and view all the answers

Approximately what percentage of eukaryotic proteins are entirely or partially intrinsically disordered?

30% Signup and view all the answers

In what way do intrinsically disordered proteins (IUPs) affect molecular interactions?

They control the affinity and dynamics of interactions via linker length. Signup and view all the answers

Which type of proteins are more commonly found in eukaryotic organisms compared to prokaryotic organisms?

Intrinsically disordered proteins Signup and view all the answers

What is the net stabilizing effect of making new hydrogen bonds during protein folding?

It has a minor stabilizing effect. Signup and view all the answers

What contributes negatively to the entropy of folding?

Loss of conformational entropy. Signup and view all the answers

How does the overall change in Gibbs free energy (G) for folding proteins generally appear?

Ranges from -20 to -60 kJ/mol, indicating slight favorability. Signup and view all the answers

What is a consequence of proteins being only marginally stable?

They have the flexibility to adapt to various functions. Signup and view all the answers

What primarily drives the favorable enthalpic contributions to protein folding?

Formation of new hydrogen bonds within the protein. Signup and view all the answers

What is primarily affecting the folding energetics when considering the entropic contributions?

The extensive range of possible extended conformations. Signup and view all the answers

Why is high stability in proteins considered detrimental to their function?

It limits their ability to adapt to different environments. Signup and view all the answers

Which factor primarily contributes to the small overall stabilizing energy of a protein?

Energy released from formed interactions. Signup and view all the answers

What is a characteristic of the molten globule state during protein folding?

Hydrophobic residues are largely buried. Signup and view all the answers

Why do proteins that fold with intermediates generally fold more slowly?

They face energetic barriers that trap them temporarily. Signup and view all the answers

What is the role of prolyl peptide isomerases in protein folding?

They catalyze the isomerization of cis to trans proline. Signup and view all the answers

What contributes to the stability of knotted proteins during folding?

Threading at least part of the chain through a loop. Signup and view all the answers

Which of the following describes a feature of molecular chaperones?

They bind exposed hydrophobic surfaces of unfolded proteins. Signup and view all the answers

What happens to a protein that forms non-native disulfide bonds?

It gets trapped and cannot properly fold. Signup and view all the answers

What characterizes the caught state of proteins during folding?

It indicates a temporary trapping due to energetic barriers. Signup and view all the answers

Which condition influences the folding of proteins in vivo compared to in vitro?

Higher levels of interacting molecules. Signup and view all the answers

What structural feature is typically observed in folding intermediates?

They have a well-defined structure but are less stable. Signup and view all the answers

How do amyloids relate to protein misfolding?

They can spontaneously refold to form stable fibrils. Signup and view all the answers

What effect does the cis-trans isomerization of proline have on protein folding?

It can become a rate-limiting step within the process. Signup and view all the answers

What is the primary role of protein disulfide isomerases?

To help break and form correct disulfide bonds in folding proteins. Signup and view all the answers

What function do intrinsically unstructured proteins (IUPs) serve in relation to proteins?

They act as flexible linkers between protein domains. Signup and view all the answers

Which statement regarding the role of the acyl carrier protein (ACP) in Fatty Acyl Synthase is accurate?

ACP transfers substrates to each catalytic site sequentially. Signup and view all the answers

What is the significance of the length of a linker in intrinsically unstructured proteins?

It controls the affinity and dynamics of interactions. Signup and view all the answers

What role do intrinsically unstructured proteins (IUPs) play in relation to membraneless organelles?

They play critical roles in protein interactions. Signup and view all the answers

How do IUPs contribute to mechanical properties in proteins?

They absorb energy like springs due to their flexibility. Signup and view all the answers

What is the primary function of the inactivation domain in K+ channels?

To block the open channel after signal generation Signup and view all the answers

How does the length of the linker sequence affect the inactivation mechanism of ion channels?

It determines the binding affinity of the inactivation peptide. Signup and view all the answers

What mechanism drives the process of phase separation in disordered proteins?

Weak self-associations based on various interactions Signup and view all the answers

Which of the following interactions is NOT typically involved in driving phase separation?

Covalent cross-linking Signup and view all the answers

How do intrinsically disordered proteins (IUPs) utilize their structure to bind to partners?

By wrapping around ordered proteins, enhancing binding surface interaction Signup and view all the answers

What is one way proteins regulate the assembly of condensates?

By modifying their phosphorylation status Signup and view all the answers

Which statement correctly describes Ddx4's behavior in cells?

It interacts selectively with ssDNA while excluding dsDNA. Signup and view all the answers

What is a unique feature of the FG repeats in the nuclear pore complex?

They self-interact to form a gel-like plug. Signup and view all the answers

What drives the specific but transient interaction of IUPs with their binding partners?

The binding energy being used to fold one of the interacting partners Signup and view all the answers

What is the significance of the N-terminus in the inactivation domain of K+ channels?

It forms the peptide that blocks the channel. Signup and view all the answers

How does phosphorylation influence the binding of IUPs?

It modifies the binding surface, increasing affinity. Signup and view all the answers

How does the use of IUPs impact cellular crowding?

They allow for smaller proteins to engage in multiple interactions. Signup and view all the answers

Which of the following accurately describes the affinity of IUPs for their targets?

Binding affinity can be in the nanomolar range with few amino acids. Signup and view all the answers

What role do small interaction driving sequences play in phase separation?

They are often interspersed within disordered regions. Signup and view all the answers

Study Notes

Trypsin Dynamics

Trypsin alternates between six distinct states in molecular dynamics simulations.
These states differ in loop conformation and substrate affinity.
The states interconvert on a timescale of 1-100 nanoseconds.
The red and green states are the most populated low and high affinity states.
The slow interconversion of these two states dominates the activation/inactivation dynamics of trypsin.

Protein Folding

A protein is considered "folded" when it is in a compact, stable, functional conformation.
Once folded, a protein primarily exists in a relatively small set of closely related conformations.
Unfolded proteins lack the interactions seen in folded structures and are less stable.
In unfolded states, proteins are not constrained by native interactions and can adopt numerous conformations, most of which are dissimilar to the native structure.

Studying Folding - Biochemically

Chemical denaturants, like urea and guanidine, destabilize protein structure by competing for favorable interactions.
Strong acids and bases destabilize by causing electrostatic repulsion.
Rapid removal of denaturants, like through dilution, induces refolding. This refolding can be monitored using biophysical techniques.

Interactions in Unfolded and Folded States

Forming a new hydrogen bond between two protein atoms during folding may not always have a net stabilizing effect.
This is because breaking existing hydrogen bonds between these atoms and water molecules requires energy.
However, burying protein groups without making hydrogen bonds is definitely destabilizing.

Protein Stability

The energetics of folding can be formulated in terms of enthalpic and entropic contributions.
The enthalpic contribution (H~~fold~~) is favorable and on the order of ~1000s of kJ/mol.
The entropic contribution (T*S~~fold~~) is also on the order of ~1000s of kJ/mol, but is destabilizing.
This is mainly due to the loss of conformational entropy.
The overall Gibbs free energy (G~~fold~~) is ~-20 to -60 kJ/mol, favorable to folding but slightly so.
The overall stabilizing energy of a protein is small compared to the energy released by the interactions formed.

Folding Energetics of Ribonuclease

The entropy of folding is strongly unfavorable.
This reflects the loss of conformational entropy as the protein is forced to adopt a single compact conformation.
The enthalpy of folding is strongly favorable.
The strongly favorable enthalpy slightly outweighs the strongly unfavorable entropy (approximately 2 hydrogen bonds worth of energy).

Marginal Stability and Function

Proteins are marginally stable, allowing flexibility for function.
They adapt to ligands, move between catalytic cycle steps, and can be modified by enzymes.
This flexibility is essential for functions like binding and catalysis.

Protein Folding Time Scale

Hydrophobic collapse and formation of local secondary structure occur rapidly (~10 ns).
Very small proteins can fold in ~1 μs.
Slow proteins can take 100s of milliseconds to fold.

Secondary Structure Formation

Collapse of a hydrophobic core and formation of secondary structure elements happen quickly, within nanoseconds.
This is driven by local interactions within the sequence.
These early secondary structure elements resemble, but are not identical to, native secondary structure and are dynamic.

Topology Acquisition

Over time, secondary structure elements rearrange and explore different topologies.
Few initial amino acid contacts resemble the native structure.
Eventually, secondary structure elements achieve a near-correct arrangement.
At this point, further improvements become cooperative, and residues rapidly lock into their correct positions.
This drives the stabilization of native topology and secondary structure elements.

Three-State Protein Folding

Some proteins go through one or more intermediate conformations before adopting the native structure.
Many proteins utilize a partially folded "molten globule" state during folding.
Other proteins form relatively long-lived (millisecond) intermediates with well-defined structures.

Folding Funnels with Intermediates

Folding intermediates can occur when proteins form structures close to the native state but slightly less stable.
These intermediates can temporarily trap proteins due to energy barriers.
Proteins that fold with intermediates typically fold more slowly.

Molten Globules

Some proteins experimentally exhibit a "molten globule" intermediate during folding.
Molten globules have most hydrophobic residues buried and much of the native secondary structure formed.
However, secondary structural elements are not always properly placed, and side chains remain unpacked.
The protein core remains dynamic, making molten globules a state before the final, cooperative folding steps.

Intermediates with Defined Structures

Some folding intermediates have well-defined structures.
These intermediates can be close enough in energy to the native structure to exist in a significant proportion (e.g., ~2%) of the protein.
Sophisticated NMR techniques can be used to determine the structure of these intermediates.

Knotting and Folding Intermediates

Some proteins have knotted topologies.
Forming a knot requires a portion of the chain to thread through a loop in another section.
This process involves interactions that are different from the final, native structure, and requires specific intermediates.
The length of the sequence that needs to thread through influences folding time.
Few proteins form knots due to the complexity of the process.

In Vivo Protein Folding

In cells, protein folding occurs as proteins are synthesized by ribosomes.
N-terminal domains in multi-domain proteins can fold before the C-terminal domains are synthesized.
In vivo protein folding in the cell can be influenced by the presence of other proteins.
In vitro protein folding occurs in dilute solutions with less interaction between proteins.

Protein Misfolding and Aggregation

While native protein structures are optimized through evolution, alternate protein conformations can exist with stable packing patterns.
These alternative packing patterns can lead to the formation of extended fibrils, known as amyloids.
Amyloids can recruit further copies of the protein, leading to a pathological phenotype.
Over 50 human diseases, including Alzheimer's disease, are associated with amyloid formation.

Alzheimer's Disease and Amyloids

The membrane adjacent region of the amyloid precursor protein (APP) is prone to self-association and amyloid formation.
This region can form various fibril structures, leading to Alzheimer's disease.
Specific mutations can promote amyloid formation.

Intrinsically Unstructured Proteins

Folding typically depends on hydrophobic collapse driven by hydrophobic residues.
However, proteins or protein regions with too few hydrophobic residues may not fold spontaneously and can remain unfolded.
These proteins are known as intrinsically disordered (or unstructured) proteins.
Despite lacking a defined structure, they play essential roles in the cell.

Functional Roles of Intrinsically Unstructured Proteins

Intrinsically unstructured proteins play critical roles in protein interactions and membraneless organelles.
They can act as linkers between protein domains, allowing flexible rearrangements.
Their length can control the affinity and dynamics of interactions.
They can act as spacers, using entropy to create a spring-like effect.
They also play roles in chaperones and stress response proteins.

Fatty Acyl Synthase and Unstructured Proteins

In fatty acyl synthase, the acyl carrier protein (ACP) is a disordered domain.
It carries substrates to catalytic sites in succession.

Ion Channel Inactivation

Ion channels can be inactivated through mechanisms involving unstructured domains.
These mechanisms enable the regulation of ion flow

Prolyl Peptide Isomerase

Proline residues can exist in cis and trans conformations.
Interconverting between these conformations requires overcoming an energy barrier.
In vivo, prolyl peptide isomerases accelerate cis-trans isomerization, allowing proteins with cis peptides to fold as fast as proteins with trans peptides.

Molecular Chaperones

Molecular chaperones assist in protein folding.
They can passively bind to exposed hydrophobic surfaces to prevent aggregation or actively unfold proteins using ATP.
They use diverse strategies for target recognition and substrate release.
Molecular chaperones are essential for proper protein folding in vivo.

Intrinsically Unstructured Proteins (IUPs)

IUPs are proteins that lack a fixed 3D structure and are highly flexible
IUPs can act as linkers between folded domains in proteins, allowing for flexible rearrangement
The length of the IUP linker can influence the strength and dynamics of interactions between domains
IUPs can act as spacers by pushing back like weak springs due to the entropic cost of restricting their conformational space
IUPs play important roles in chaperones and stress responses
IUPs have critical roles in protein interactions and the formation of membraneless organelles

IUPs in Fatty Acyl Synthase (FAS)

The acyl carrier protein (ACP) in FAS is a domain that carries the substrate (fatty acid, FA) to the various catalytic sites in the enzyme
The FA is attached to the ACP via a phosphopantheinate group
The ACP moves between the active sites within FAS, ensuring the correct sequence of reactions and efficient FA synthesis

IUPs in Ion Channel Inactivation

Ion channels, like the K+ channel, are rapidly inactivated after activation, ensuring a short burst of signal
The inactivation process relies on an inbuilt inactivation domain within the channel that specifically blocks the open channel
In the ball and chain model of inactivation, the N-terminus of the channel protein forms the inactivation domain
This domain is a short peptide (~20 amino acids) that binds within the ion channel, inhibiting its activity
The inactivation peptide is attached to the transmembrane domains via a longer linker sequence (~50 amino acids)
This linker is disordered and tethers the inactivation peptide near its target
The linker length influences the affinity of the inactivation peptide for its target, as longer linkers allow for greater diffusion and a longer search time for the binding site
This system allows for tuning the inactivation timing by simply adjusting the length of the linker sequence.

IUPs in Phase Separation

A special group of IUPs can form multiple weak, yet favorable, self-associations
Under appropriate conditions, these IUPs can form separated droplets within the solution, a process called phase separation
The resulting condensates can recruit other molecules, such as proteins and nucleic acids, that interact favorably with the condensate
These interactions can involve hydrophobic interactions, cation-pi interactions, or favorable electrostatic interactions
Sequence is crucial for condensate formation, as scrambled versions of such proteins do not condense
Condensates can also involve specific interactions between protein domains or between proteins and RNA
Covalent modifications can regulate the assembly and disassembly of these protein condensates.
Other proteins and nucleic acids can selectively partition into these localized regions, leading to concentration of specific components
This concentration can promote processes like transcription, signaling events, or concentrate factors for molecular processing.
Examples of condensates include nucleoli, stress granules, and viral replication condensates, such as those associated with COVID-19.
Condensates are crucial for cellular organization and directly impact many biological processes, especially signaling.

Examples of IUPs in Phase Separation

Ddx4: an IDP that forms phase-separated droplets both in vitro and in cells
Ddx4 interacts with itself through regions of negatively and positively charged residues.
Ddx4 droplets preferentially sequester single-stranded DNA (ssDNA), while excluding double-stranded DNA (dsDNA).
Methylation of arginine residues in Ddx4 can block self-association, interfering with cation-pi interactions.
FG repeats in the nuclear pore complex (NPC): extended domains containing a repeated sequence with a Phe-Gly motif
These FG motifs self-interact, forming an agarose-like gel when expressed recombinantly
This gel-like structure acts as a self-interacting plug, preventing proteins that are excluded from the nucleus from passing through the NPC.

IUPs in Disorder-to-Order Transitions for Binding

IUPs are common in signaling proteins, often binding to already folded partners
The IUP can wrap around its target protein, allowing interactions with a large number of amino acids and achieving high affinity with a relatively small number of interacting residues
Binding often depends on specific modifications, like phosphorylation, allowing transmission of a signal
An example is the TAZ-1 protein (pink), which becomes structured upon binding.
Binding an IUP to a pre-ordered domain allows for more efficient use of the available binding surface
The IUP can wrap around its interacting partner, utilizing a much larger surface area
This allows for the interacting partner protein to be smaller than if it were binding another globular protein
The IUP itself can be short (a few tens of amino acids) as it does not need to form a hydrophobic core, making most of its residues available for interaction
This leads to smaller interacting proteins, reducing crowding within the cell and allowing for a more compact genome.

HIF-1α Binding to TAZ-1

HIF-1α (IUP) binds to the TAZ-1 domain of CBP (a pre-ordered domain)
HIF-1α forms three α-helices upon binding, which were not present in the unbound form
This complex buries a large surface area, leading to a very tight (KD 7 nM) and specific interaction
The binding is enthalpically driven, with highly unfavorable entropy of binding, consistent with the ordering of HIF-1α upon binding

Using IUPs for Highly Specific, Transient Interactions

Tight protein-protein interactions often imply high specificity, but also slow dissociation rates
To achieve both specificity and transient interactions, energy gained from the interaction can be diverted into folding one of the partners
This leads to a more dynamic binding event, which is important for signaling pathways
In the case of HIF-1α binding to TAZ-1, the binding energy is used to fold HIF-1α, contributing to the transient nature of the interaction

Competition for Binding Sites

A single scaffold protein like TAZ-1 can recognize multiple dissimilar IUP targets
CITED2 competes with HIF-1α for binding to TAZ-1, antagonizing HIF-1α's action
While CITED2 and HIF-1α bind the same site, they use different structural motifs and bind with their termini in different locations
CITED2 binds to TAZ-1 with a higher affinity than HIF-1α (~33x), allowing it to outcompete HIF-1α and shut down the hypoxic response.

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