Protein Folding, Missfolding and Degradation

Lecture 4 September 4th

Motif

• combination of two or more secondary structures that form distinct 3D structure found in multiple proteins

  • is associated to a particular function

• smaller than a domain

Domains

• a distinct region of the protein structure

• can represent a particular function, structure or refer to the spatial relationship with the rest of the protein

unstructured regions (in DNA)

• regions in DNA without a fixed structure

• potentially more flexible 

  • this is in addition to the regions with the secondary structure

  • this is also the case in most proteins

how may domains does DNA contain ? what can we say about the motif(s) in each

• 4 domains 

• each can contain multiple motifs

Domain IV (DNA) characteristics

• Mediates DNA binding

• contains two motifs that allow the interaction w specific seqs on DNA

  • a helix-turn-helix

  • a basic loop

Domain III (DNA) characteristics

• Mediates AAA ATPase

• contains motifs for binding and hydrolysis of ATP

  • walker A

  • walker B

• contains also other motifs and multiple other functions

what is the hierarchy of protein structure (explained through protein folding) 

1. polypeptide folds regions w secondary structure

2. secondary structure elements grp into motifs

3. … then into domains

4. finally tertiary structure is acquired 

how can each AA residue rotate on its axis

• at aa residue portion of the polypeptide, axis of the backbone can rotate at the bonds connecting the alpha Carbon (Calpha) to the carbonyl and amide group

  • rotation is still limited by steric constraints imposed bythe backbone and side chains

why does proline confer special conformation to proteins ?

• because 5-7% of the peptide bonds with proline acquire a cis-configuration

  • Proline is the only naturally occurring amino acid that can exist in both cis and trans conformations

which configuration do most proteins favor and what does it result in

trans configuration (relative to the peptide backbone)

=> results in a linear backbone

trans configuration

functional groups on opposite side of plane

cis configuration

functional groups on same side of plane

isomerization

chem process by which a compound is transformed into any of its isomeric forms

Can isomerization between cis and trans proteins occur spontaneously? 

yes but it is slow

Peptidyl-proline isomerases (PPlases)

enzymes that speed up the process during folding

what are results of isomerization of a single proline

can dramatically change protein structure and affect its activity

What does the combination of many small changes in the orientation of proteins despite the limitations in rotation at each aa residue?

proteins can twist and turn

how long is the process of folding?

it is very rapid — micro s to milli s

can proteins fold on their own? what would this indicate

yes some can (test tube experiments)

demonstrates that info for structure is in the protein sequence

how are hydrophobic AAs arranged in a properly folded protein

they are burried in the core and not exposed at the surface

what are hydrophobic patches at protein surface a sign of

they are a sign of misfolding

chapeones

• proteins that help guide protein folding along productive pathways by permitting partially misfolded proteins to return to proper folding pathway 

• they facilitate the folding of many proteins

• they recognize exposed hydrophobic residues

what are 3 functions of chaperones

• can fold newly made proteins

• refold misfolded or unfolded proteins 

• dissemble potentially toxic protein aggregates that form due to protein misfolding

how do chaperones work

they work through ATP-dependent cycles of binding to and release from misfolded “client” molecules at exposed hydrophobic patches

why is it useful that chaperones block the hydrophobic patches on “client” proteins?

it keeps the folding/refolding protein isolated while productive folding events occur

what are the two types of of chaperones

• chaperones

• chaperonins

Hsp70 (Heat-shock Proteins) chaperone mechanism

• binds to short segments of an unfolded protein (like those newly syntheiszed emerging from ribosome)

• binding & hydrolysis of ATP results in conformation change of Hsp70 that are needed for it to function 

  • i.e. for protein folding assistance

what are chaperonins?

much larger complexes than chaperones that isolate unfolded proteins

GroEL chaperonin example

• composed of two stacked rings

  • each composed of 7 sub-units (proteins)

• each ring interacts with a seren-subunit co-chaperone that actis liek a lid (this is GroES which also contains 7 subunits)

• in center GroEL there are chambers where all or part of protein enters

  • proteins less than 60kDa in mass are captured by hydrophobic residues near the entrance of the chamber

• ATP hydrolysis regulates th’e cycle

• Protein folding in the two rings is coordinated

  • multiple cycles can be recquired for proper folding

what happens to irretrievably misfolded proteins?

they are marked for degradation

what is the system for protein degradation?

ubiquitin/proteasome

• Poly ubiquitin '“tags” damaged or misfolded proteins for degradation

Ubiquitin

• a small protein

  • 78aa residues

  • 8.6 kDa

• what tags proteins for degradation

ubiquitylation

• ubiquitin becomes covalently linked to the lysine residue of target proteins

  • they always bind to lysine

• to mark protein for degradation

what are the steps and machinery to covalently link ubiquitin to other proteins? 

1. a carboxyl terminus is activated 

2. ubiquitin transferred to a ubiquitin conjugating subunit: E3 ubiquitin ligase

• there are 600 E3 coded in our genomes

  • each w specific substrate binding

polyubiquitinynation mechanism

1. ubiquitin ligases recognize exposed hydrophobic residues

2. they then add multiple ubiquitins to protein (forming chain of 4 or + ubiquitins)

3. Polyubiquitinynated proteins are recognized by Ub receptors in the proteasome

4. Deubiquitinases (Dubs) hydrolyze bonds between ubiquitins to recycle them

5. ATPase driven auxiliary proteins unfold proteins and transports the core for degradation

6. once inside inner chamber, polypeptides digested into short fragmetns of 2-24aa in length

7. Peptidebinds of hydrophobic,acidic and basic residues are cleaved at the active site 

8. resulting peptides further degraded into single AAs in cytoplasm

What is a proteasome

It is a large protein complex, like a cellular machine, that breaks down unwanted or damaged proteins into smaller fragments. It is crucial for maintaining cellular health and homeostasis by eliminating proteins that are no longer needed, such as those that are misfolded or aggregated.

What does the accumulation of misfolded proteins lead to

protein aggregation

Protein aggregation

when misfolded proteins or incompletely degraded proteins iteract with each other

• hiding their hydrophobic residues

• forming aggregates

what leads to the formation of aggreates

• high protein concentration

• changes in environmental conditions

what are two ways protein aggregates can be?

• amorphous

• well-organized

  • i.e. amyloid state

IRL examples of protein aggregation

• egg white contains albumin

• milk contains casein

how can we see that proteon aggregation occurs in cells?

green fluorescent spot shows position of aggregates

—> we see appearance of spots in some cells as they grow suggesting the accumulation of protein aggregates

how are amyloid fibrils formed

• they are formed by the generation of short segments (6-12 aa residues in length) that form long arrays or filaments of beta-sheets

  • each beta strand is oriented perpendicularly to the axis of the filament — two stacks twist about one another => forming photofilaments => many of the forming fibrils

where can we find amyloids and what are they markers for

• they can be found in tissues

• they are markers for disease

  • amyloid formations are associated to age but is is also more prevalent in mutant proteins

examples of neurodegenerative diseases that contain amyloids

• alzheimers

• parkinsons

• the transmissible spongiform encephalopathy (aka “mad cow disease”)