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Flashcards in Protein Folding Deck (28)

Basic stabilization in Protein Folding

Secondary- constrained by partial double bond character of the peptide bond (no rotation around alpha carbon)

Tertiary- stabilized by weak no covalent bonds between regions of the polypeptide (H bonds, electrostatic attraction, van Der Waals, hydrophobic interactions)


Basic protein folding mechanism

Folding occurs spontaneously

Nascent polypeptide chain

-folding and cofactor binding (non-covalent interactions)

-covalent modification by glycosylation, phosphorylation, acetylation (attach these groups onto the amino groups of the AA)

-binding to other protein subunits

Mature functional protein


Ligand Binding

Proteins can bind specifically to other substances including other proteins, small organic molecules, or ions

Strong interactions causing proteins to assume different conformation and biological activity

Brought to close proximity during folding

Many drugs act as ligands and become biologically active when bound to specific proteins


In order for a protein to be biologically active it must..

1) fold correctly
2) bind appropriate ligands
3) be co- or posttranslationally modified
--> phosphorylation w/ specific protein kinases
--> glycosylation


What happens to proteins destined for secretion?

They do not fold co-translationally and require the participation of a class of proteins called molecular chaperones


Chaperones- an overview

Proteins that bind unfolded, or partially folded, proteins and prevent them from aggregating to associating with other proteins until native conformation is assumed

Prevent hydrophobic patches from associating with each other

They DO NOT change the thermodynamics of the folding process

They restrict folding pathway such that # of possible intermediate states is limited --> folding becomes faster


Chaperones- Hsp60 and Hsp70

Both have ATPase activity and preferentially bind unfolded proteins in the ADP state

ADP exchanged with ATP forming an ATP-chaperone complex which is released from sections of correctly folded proteins

Binding and release of chaperone proteins and ADP-ATP exchange is repeated until native conformation is reached

Hsp70 --> specifically recognize hydrophobic regions on the surface of unfolded proteins even before protein leaves ribosome

Hsp60 --> does not function co-translationally; assumes barrel like structure that binds fully synthesized proteins
^also called chaperonin--> isolates newly synthesized unfolded or misfolded protein preventing aggregation
^found in the mitochondria


Hsp70 Examples in both Prokaryote and Eukaryotes

Prokaryote- DnaK

Eukaryotes- Hsc73 (cytosol)
BiP (ER)


Hsp60 Examples in both Prokaryotes and Eukaryotes

Prokaryote- GroEL

Eukaryotes- TriC (cytosol)
Hsp60 (mitochondria)
Cpn60 (chloroplasts)



Large abundant protein complexes that contain proteases which degrade abnormal proteins

Central hollow core (20S proteasome)
1 or 2 19s protease caps (Hexamer)


Proteasome- the Core

Core --> multiple subunits with 4 heptameric rings some of which are proteolytic enzymes with different specificities arranged so that the active sites line the hollow core

The proteases cleave target proteins into free amino acids and very small peptides (further degraded in cytoplasm) --> degrades in 7-8 AA peptides

Highly processive


Types of Proteolytic Enzymes

Chymotrypsin- likes the cleave after hydrophobic amino acids (aromatic?)

Trypsin- likes to cleave after basic amino acids

Peptidylglutamyl- likes to cleave after glutamate residues


Proteasome- the 19s Cap

19s cap --> ends of the core and contain ring with 6 protein subunits

^misfolded proteins threaded thru ring to core for degradation (ATP dependent)

^As target proteins are threaded thru the caps, they are unfolded in a reaction driven by ATP hydrolysis

Also have regulatory role--> recognize misfolded proteins targeted for degradation by ubiquitination



Ubiquitin- single chain polypeptide (76 AA) that signals for degradation --> contains hydrophobic globular core

3 Enzyme Reaction

E1- ubiquitin activating enzyme
E2/E3- ubiquitin ligase

1) C terminus of ubiquitin attached to E1 through a thioester bond --> ATP to AMP (use of 2 phosphates in this reaction)
2) activated ubiquitin transferred to E2 with release of E1
3) E2/E3 complex attaches ubiquitin to the amino group of lysine in a target protein thru an isopeptide bond
4) Addition of other molecules of ubiquitin occur by linkage of a glycine residue in ubiquitin 2 to lysine 48 in ubiquitin 1 and so on to form linear chain

Different E2/E3 complexes in different cells

4 residues of ubiquitin needed for targeting to proteasome



Protein Disulfide Isomerase
-found in ER
-promotes formation of disulfide bonds
-catalyzes oxidation of SH group on cysteines to form disulfide bonds


Why don't disulfide bonds form in cytoplasmic proteins?

Due to the reducing environment of the compartment

Extracellular regions and lumen of organelles --> have disulfide bonded proteins



Binding Protein
-chaperone protein
-member of Hsp70
-hydrolyzes ATP as it guides the transport of fully synthesized proteins into the ER through the translocon on the ER membrane
-recognizes misfolded proteins by binding to an aberrant series of amino acids on the surface of the protein that should've been on the inside

-misfolded proteins with bound BiP are retained in ER and cannot pass to the Golgi


N-glycosylation...what's the role of the Lectins?

N-linked glycans are added to specific Asn residues as a complex oligosaccharide containing N-acetylglucosamine, mannose and 3 terminal glucose residues

NORMAL--> all 3 glucose residues and 1 or more mannose residues are removed in the ER with further trimming in the Golgi
2) C&C bind glycoproteins that have been processed by glycosidases to remove 2 of the 3 glucose residues leaving the incompletely processed protein with 1 glucose residue
3) if 3rd glucose is removed, the protein is recognized as fully processes and C&C dissociate allowing protein to pass onto Golgi


What is the role of glucosyl transferase?

Improperly folded proteins can act as substrates for glycosidase BUT are immediately acted upon by GT that adds back the terminal glucose residue

-does not recognize fully folded proteins

-C&C remain bound to incompletely folded proteins through the cycle of glucose removal and addition and only dissociated when fully folded protein is not recognized by GT



If protein never achieves native conformation in ER --> must be eliminated

Proteins transferred from ER back to cytoplasm by this process

1) protein threaded thru same translocon used in co-translational import (ATP needed)
2) mediated by ER and cytoplasmic chaperones
3) In cytoplasm --> proteins deglycosylated, ubiquinated, and targeted for degradation



Unfolded Protein Response
Triggered by accumulation of misfolded proteins in ER

--> UPR triggers several intracellular signaling mechanisms which activate pathways to increase protein folding capacity in ER (and reduce level of misfolded proteins)

1) Translation is inhibited (PERK pathway)
2) expressions of genes for chaperones upregulated (ATF6 pathway)
3) expressions of genes for protein degrading enzymes upregulated

*APOPTOSIS if misfolded proteins keep accumulating*


UPR Continued

Low BiP means lots of BiP has attached to unfolded proteins --> initiation of IRE1 pathway --> regulated mRNA splicing

PERK pathway --> lowers protein synthesis to slow process down by selective translation

ATF6 pathway --> increase transcription of chaperones



Balance of protein synthesis, folding, and degradation (protein homeostasis)


Factors that contribute to increased protein misfolding

Over production
Impaired degradation
Heat shock
Absence of binding partners
Alterations in post translational processing
Oxidative stress


Misfolding of a protein can lead to 2 things:

Dominant inheritance- gain of toxic function
Recessive inheritance- loss of biological function


Amyloid Fibrils

Can be pathogenic

Beta strands perpendicular to fiber axis
Beta sheets parallel to fiber axis



Misfolded proteins in the cytoplasm are targeted to specific intracellular inclusions

Destination depends on ubiquitination status and transport depends on cytoskeleton

JUNQ- protein is degraded and are mobile (usually have ubiquitinated proteins)

IPOD- proteins persist and are trapped (non ubiquitinated)

^^these are mechanisms for sequestering misfolded proteins so that they cannot interact with normal proteins


Why does the UPR increase the amount the lipids in the cell along with the chaperones?

Majority of the unfolded proteins coming into this pathway are from the membrane --> more membrane proteins needed --> now more membrane needed itself --> need more lipids for the structure