4.4 Protein Denaturation and Folding

How do organic solvents denature proteins?

Disrupt the hydrophobic interactions that make up the stable core of globular proteins.

What happens while larger proteins with multiple domains are synthesized?

Domains near the amino terminus (which are synth. first) may fold before the entire polypeptide has been assembled.

How is protein folding sometimes facilitated?

-Hsp70 chaperones -chaperonins -Disulfide bond formation and the cis-trans isomerization of Pro peptide bonds are catalyzed by specific enzymes.

Describe the hierarchy of protein folding in cells?

-Initially, regions of secondary structure may form -This is followed by folding into motifs and domains -Large ensembles of folding intermediates are rapidly brought to a single native conformation.

What does the spontaneous renaturation of some denatured proteins show?

-It shows that the tertiary structure is determined by amino acid sequence.

Char. of Hsp70 (heat shock proteins, MW 70k) family

-MW near 70k -More abundant in cells stressed by elevated temps. -bind to regions of unfolded polypeptides that are rich in HYDROPHOBIC residues. -"protect" both proteins subject to denatured. by heat and new peptide molecules being synth (and not yet folded) -block folding of proteins that must remain unfolded until they're translocated across a membrane. -facilitate quaternary assembly of oligomeric proteins -bind and release pp in cycle that uses energy from ATP hydrolysis and involves other proteins.

What is proteostasis and what does it involve?

-Proteostasis is the maintenance of the steady-state collection of active cellular proteins required under a particular set of conditions. -Proteostasis involves a set of pathways and processes that fold, refold, and degrade polypeptide chains.

What demonstrates the relationship between structure and function?

-The 3-D structure and the function of most proteins can be destroyed by denaturation.

Difference between GroES lid in chaperonins and Hsp60 system in eukaryotes?

-The Hsp60 system is similar, but instead of the GroES lid, protrusions from the apical domains of the subunits flex and close over the chamber. -The ATP hydrolytic cycle is also slower in the Hsp60 complexes, giving the constrained proteins inside more time to fold.

Under what circumstances can unfolded proteins accumulate in eukaryotes and what do these circumstances trigger?o

-When stress conditions arise -When protein synthesis threatens to overwhelm the protein-folding capacity of the ER -Triggers the unfolded protein response (UPR)

Binding of an unfolded pp by an Hsp70 could...?

-break up protein aggregate or prevent formation of a new one -May deliver to a chaperoning

Cystic Fibrosis?

-caused by defects in a membrane bound protein called cystic fibrosis transmembrane conductance regulator (CFTR). -CFTR acts as a channel for Cl- ions

What does the loss of protein structure lead to? (paragraph header)

Loss of function

What do regions of low stability allow proteins to do?

May allow a protein to alter its conformation between 2 or more states.

Why can many proteins take on the amyloid fibril structure as an alternative to their normal folded conformations?

Most of these proteins have a concentration of aromatic amino acid residues in a core region of B sheet or alpha helix. -Proteins secreted in an incompletely folded conformation. -The core (or some of it) folds into a Beta sheet BEFORE the rest of the protein folds correctly. -The Beta sheets from two or more incompletely folded protein molecules associate to form an amyloid fibril.

What must proteins maintain to function?

Most proteins must maintain conformational flexibility to function.

Serum amyloid A (SAA) protein?

-deposits in connective tissue of spleen, kidney, liver, and around the heart.

Other methods of denaturation?

-extremes of pH -miscible organic solvents like alcohol or acetone -solutes such as urea, guanidine hydrochloride -detergents ALL unique in that no covalent bonds in the polypeptide chain are broken.

Symptoms of primary systemic amyloidosis? Most affected organs?

-fatigue, hoarseness, swelling, weight loss, death w/in first year of diagnosis -Kidneys and heart are most affected.

Effects of disease related mutations in collagen?

-mutations also cause defective protein folding.

Chaperonins?

-required for folding of non-spontaneously folding proteins. -analogous chaperoning system in eukaryotes is Hsp60. -some necessary for bacterial viruses

What percentage of polypeptides synthesized in all cells are destroyed because they didn't fold properly?

1/4

Protein disulfide isomerase (PDI)

1st enzyme discussed, it catalyzes the interchange, or shuffling, of disulfide bonds until the bonds of the native conformation are formed.

Overall structural features?

2 layers of Beta sheet

2nd step in chaperonin formation?

2. The protein is then trapped within the chamber when it's capped transiently by the GroES "lid"

Peptide propel cis-trans isomerase (PPI)

2nd enzyme discussed, catalyzes the interconversion of the cis and trans isomers of Pro residue peptide bonds, which can be a slow step in the folding of proteins that contain some Pro peptide bonds in the cis conformation

3rd step in chaperonin formation?

3. GroEL undergoes substantial conformational changes, coupled to slow ATP hydrolysis, which also regulates the binding and release of GroES.

What happens to symptoms of Parkinson and Huntington disease symptoms if the expression of the Hsp70 chaperone is increased?

Symptoms are highly suppressed.

Define proteostasis

The continual maintenance of the active set of cellular proteins required under a given set of conditions

How does the amyloid fibril affect the folding of the other parts of the protein?

The other parts fold differently and remain on the outside of the Beta sheet core in the growing fibril..

What happens after denaturalization and removal of urea and the reducing agent?

The randomly coiled, denatured ribonuclease spontaneously refolds into its correct tertiary structure, with FULL restoration of its catalytic activity. Even the 4 disulfide bonds are reformed in the same position as the native ribonuclease.

Autophagy

amyloid aggregates that formed before the UPR was initiated are removed by; -First, encapsulation in a membrane -And second, contents vessel degraded following vesicular docking with a cytosolic lysosome.

What are the different misfolded protein degradation systems? Effects of defects in these systems?

-Autophagy -Ubiquitin-proteosome system -decreases capacity to deal with misfolded proteins -Increases propensity for the development of amyloid-related diseases.

Secondary systemic amyloidosis

-Chronic infectious or inflammatory diseases such as rheumatoid arthritis, tuberculosis, cystic fibrosis, and some cancers. Sharp increase in secretion of an amyloid-prone polypeptide called serum amyloid A (SAA) protein.

Most common cystic fibrosis-causing mutation? Effects of mutation?

-Deletion of a She residue at position 508 in CFTR -Deletion causes improper protein folding. Most of protein is degraded and its normal function lost.

Causes of primary systemic amyloidosis?

-Deposition of fibrils consisting of misfolded immunoglobulin light chains, or fragments of light chains derived from proteolytic degradation.

Neurodegeneration

-Extracellular amyloid deposition-Alzheimer's -Intracellular aggregation involving tau protein-Dementia and parkinsonism

1st step in chaperonin formation?

1. An unfolded protein bound to an exposed hydrophobic surface near the apical end of one GroEL chamber.

Hierarchy of major folding pathways? (2) Describe what's involved in each step.

1. Local secondary structures form first -Certain aa sequences fold into alpha helices or beta sheets readily, guided by constraints of secondary structure. -Ionic interactions, involving charged groups near one another in the linear sequence of the pp chain play role in guiding these early folding steps. 2. Long-range interactions between secondary structure elements that come together to form stable, folded structures. - Hydrophobic interactions play key role in this process as aggregation of non polar aa side chains provide an "entropic" stabilization to intermediates and the eventual final folded structure. -This 2nd process continues until complete domains form and the entire polypeptide is folded.

What three kinds of processes contribute to proteostasis?

1. Proteins are synthesized on a ribosome. 2. Multiple pathways contribute to protein folding, many of which involve the activity of complexes called "chaperones." 3. Proteins that are irreversibly unfolded are subject to sequestration and degradation by several additional pathways.

define denaturation

A loss of 3-dimensional structure sufficient to cause loss of function

How does urea denature proteins?

Also disrupts H-bonds

Amyloidoses? Examples and mechanism

Amyloidoses-refers to the diseases associated with a misfolding mechanism in which a soluble protein that is normally secreted from the cell is secreted in a misfiled state, and converted into an insoluble extracellular amyloid fiber. Examples include; type 2 diabetes, Alzheimer's, Huntington disease, and Parkinson disease.

Ubiquitin-proteasome system

Another misfolded protein degradation system

What is the effect of aromatic residues on amyloid structure?

Aromatic side chains stabilize the amyloid structure significantly

Why are some amyloid diseases associated with particular organs?

Because the amyloid-prone protein is generally secreted only by the affected tissue, and its locally high concentration leads to amyloid deposition around that tissue.

How is a globular protein's tertiary structure determined?

By it's amino acid sequence

Function of PDI?

Catalyzes the elimination of folding intermediates with inappropriate disulfide cross-links.

Which processes occur fastest?

Close-range interactions (b/w pairs of res. generally near each other in the seq.) tend to fold FASTER than proteins with more complex folding patterns and many long-range interactions.

How does heat effect protein structure?

Complex effects on many weak interaction in a protein, primarily on the H-bonds.

What are 2 benefits of a protein being constrained?

Constraining a protein in the chamber; A) prevents inappropriate protein aggregation. B) restricts the conformational space that a polypeptide chain can explore as it folds.

Why are regional variations in stability essential?

Essential for protein function

What did the Anfinsen experiment show?

Gave first evidence that the amino acid sequence of a polypeptide chain contains all the information required to fold the chain into its native, 3-D structure. Only a minority of proteins, mostly small and inherently stable, will fold spontaneously into their native form.

2 major families of chaperones?

Hsp70 and the chaperonins

Protein huntingtin

Huntington disease-involves huntington protein w/ long polyglutamine repeat. Sometimes longer than normal, more subtle type of intracellular aggregation occurs.

In eukaryotes, where do proteins destined for secretion undergo their initial folding?

In the endoplasmic reticulum

Where does the amyloid fibril grow?

In the extracellular space

What can improper protein folding lead to?

Often improperly folded proteins have exposed hydrophobic surfaces that render them "sticky" and lead to the formation of inactive aggregates.

Lewy bodies

Parkinson disease-misfolded protein alpha-synuclein aggregates into spherical filamentous masses called Lewy bodies.

Why is proteostasis important?

Partially unfolded proteins and protein-folding intermediates that escape the quality-control activities of the chaperones and degradative pathways may aggregate, forming both disordered aggregates and ordered amyloid-like aggregates that contribute to DISEASE and AGING processes.

How are the B-sheets oriented?

Perpendicular to the axis of the fiber

Levinthals paradox?

Protein folding is NOT a completely random, trial and error process. There must be shortcuts.

What is the molecular basis of many human diseases including the amyloidoses?

Protein misfolding

What happen's to a protein's conformation as temperature is increased slowly?

Protein's conformation generally remains intact until an abrupt loss of structure (and function) occurs over a narrow temperature range.

What happens inside the chamber?

Proteins inside the chamber has approx. 10 seconds to fold-the time required for the bound ATP to hydrolyze.

Requirements for proteostasis? (3)

Proteostasis requires; 1. The coordinated function of pathways for protein synthesis and folding. 2. The refolding of proteins that are already partially unfolded. 3. The sequestration and degradation of proteins that have been irreversibly unfolded.

Describe the denaturalization of ribonuclease A by a urea solution in the presence of a reducing agent.

Reducing agent cleaves the 4 disulfide bonds to yield 8 Cys residues, and the urea disrupts the stabilizing hydrophobic interactions, thus freeing the entire polypeptide from its folded conformation.

Last folding pathway discussed?

Some proteins require 2 enzymes (PDI and PPI) that catalyze isomerization reactions

Chaperonin structure?

Structured as a series of multisubunit rings, forming a two chambers oriented back to back.

How do the two chambers in a GroEL coordinate?

They alternate in binding and releasing unfolded polypeptide substrates.

Describe the thermodynamics of protein folding and as a free-energy funnel as folding occurs.

Unfolded states are characterized. by high degree of conformational entropy and high free energy. As folding proceeds, narrowing of funnel reflects the DECREASE in conformational space to be searched as the protein approaches native state. (Delta G represents depth, native state (N) is at the bottom bc it's the lowest free energy point). The # and types of folding intermediates determines the shape of funnel. Folding intermediate w/ sig. stability and a finite lifetime is represented as a "local free-energy minimum," or a depression on the surface of the funnel.

What does the abruptness of protein change suggest?

Unfolding is a cooperative process: loss of structure in one part of the protein destabilizes other parts.

When is the protein released?

When the GroES cap dissociates but can rebound rapidly for another round if folding hasn't been completed.

How do extreme pH's denature proteins?

alter the net charge on the protein, which causes electrostatic repulsion and the disruption of some H-bonding.

Unfolded Protein Response (UPR)

constituted by a set of transcriptional regulators that bring various systems into alignment by increasing the concentration of chaperones in the ER OR decreasing the rate of overall protein synthesis, OR BOTH.

Amyloid fibers

fibers are highly ordered and unbranched, w/ a diameter of 7-10nm and a high degree of B-sheet structure.

What do chaperones do?

help proteins being synthesized on ribosomes fold into their native conformations. Many also function to refold proteins that become transiently unfolded.

What do denatured proteins exist as under most conditions?

partially folded states

What does denaturation often lead to?

protein precipitation, a consequence of protein aggregate formation as exposed hydrophobic surfaces associate. Aggregates are usually highly disordered. E.g. the protein precipitate that is seen after boiling an egg white is one example. Some ordered aggregates are also observed

Chaperones

proteins that interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways or providing microenvironment in which folding can occur

define renaturation

the process by which certain denatured (by heat, pH extremes, or denaturing reagents) globular proteins regain their native structure and their biological activity when returned to conditions in which their native conformation is stable.

Why are inactive aggregates bad for cells?

they may not perform their normal function, but they are NOT INERT. Their aggregation in cells lie at the heart of diseases ranging from diabetes to Parkinson's and Alzheimer's diseases.