Protein Folding Flashcards
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)
Proteasomes
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
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
PDI
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
BiP
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
Retrotranslocation
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
UPR
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
Proteostasis
Balance of protein synthesis, folding, and degradation (protein homeostasis)
Factors that contribute to increased protein misfolding
Over production Impaired degradation Mutations Heat shock Absence of binding partners Alterations in post translational processing Oxidative stress Aging
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
JUNQ and IPOD
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
