Protein folding and misfolding Flashcards
Levinthal’s Paradox
it only takes a few seconds
discrepancy from calculation which is thought to be 10^48
proteins dont expolre all the potential conformations open to it
therefore protein folding cannot be a completely random process
there must be folding pathways
protein folding in the cell
in the ribosome
synthesis from amino terminus to carboxy terminus using mRNA template
2d structure as polypeptide chain grows
how many possible conformations are there for a polypeptide of 100 residues
3^100
assume each residue can adopt any one of 3 conformation
protein denaturation to study protein folding
in the lab urea or guanidine hydrochloride are used as denaturants if not heat
guanidine more so than urea as it has an oxygen making it a good acceptor for hydrogen bonds therefore are able to compete with the with the hydrogen bonds that stabilise the protein
hydrophobic core disrupted too
disulphide bonds are reduced with beta mercapteothanol
extent of the unfolding is followed bio physically by a uv spectroscopy
Anfinsen’s ribonuclease experiment
denatured ribonucelease A (enzyme which degrades RNA) with urea and beta-mercoptoethanol (BME)
refold by removing urea and BME by dialysis
put the protein in cellophane bag as semi permeable membrane
proteins held back and urea or BME fall out
native fold regained
tried both orders of removal = misfolded state = inactive
what did Anfinsen concluse
conc = loss of native conformation leads to loss of activity so the native conformation of a protein is required for catalytic activity
conc 2:
an unstructured inactive polypeptide can fold into an active protein so
the native conformation of. a protein is determined by its primary structure
conc 3: the native structure forms spontaneously upon removal of the denaturant so the native conformation of a protein is its lowest energy state
the free energy funnel
the thermodynamics of protein folding can be represented as a funnel
semi stable folding intermediates in local energy minima get stuck misfolded and never gets to the lower energy state of the origin
what is the pathway beween the intermediates
Hierarchical folding
secondary structure forms first
followed by a hyrophobic collapse an formation of the teriary structure
folding viz a molten globule
initiated by a hydrophobic collapse and followed by secondary an tertiary structure formation
probably fold by multiple complex pathways that involveboth of the above
there is no single universal pathway of protein folding
protein folding in vivo
proteins fold spontaneously as they are synthesised
others are incapable of folding in isolation regardless of the pathway taken
get trapped in partially folded conformations that are susceptible to aggregation which may lead to cell death and disease
Molecular chaperones
to avoid aggregation folding of many proteins is facilitated by the action of specialised proteins
interact with partialy folded polypeptides
faciliating correct pathways by providing microenvironments for folding to occur
HSP chaperones Heat Shock Protei protect proteins that have been denatured by heat and polypeptides that are being synthesised
HSP70 and HSP40
- DNAJ binds to the unfolded or partially folded protein and then to dNA K
2 DNA J stimulates ATP hydrolysis by DNAK-aDP binds tightly to the unfolded protien
ATP binds to DNAK and the protein dissociates
Although they use ATP do not actively promote protein folding they just provide a safe haven for unfolded polypeptides
Chaperonins: GroEL/GroES
proteins that fail to fold in the Hsp70.HSP40 system are passed onto the second class of chaperones
unfolded protein are bound within packets of the GroEL complex and the pockets are capped transiently by the GroEL ‘lid’
ATP hydrolysis is linked to conformational changes within GroEL-GroES
an internal ring of hydrophobic residues which interact with unfolded proteins inticing them into the cavity
the lower GroEL heptomer has undergone a majjor conformational change
hides ring of hydrophobic residues allowing protein folind inside its large internal cavity
The hydrophobic residues become consumed in part because of the binding of this cap protein, which gives the whole molecule the shape of a bullet. And so the polypeptide chains released inside the cavity.
The protein coming out can be fully folded, but more often than not, it is partially folded but committed to adopt native confirmation. particularly encouraged by being trapped in this cage where it can’t aggregate with all the molecules of the same type.
Peptide Prolyl cis-trans Isomerase (PPI)
cis trans 1000* less stable than trans form except in proline there is only a 4 fold difference
in proteins peptide bonds are mainly trans
but in proline amino acid the energ barriers between cis and trans peptide bonds re solfter so in a random coil tconformation about 10% of peptide bonds between and amino acids and proline are cis
role of PPT is a configurational isomerase protein which interconverts cis and trans forms of X-Pro dipeptides
without PPI cis-trans isomerisation is the rate limiting step in protein folding
Protein Disulphide Isomerase (PDI)
Configurational isomerase that catalyses exchange of disulphide bonds within proteins
removes folding intermediates that have incorrect disulphide bonds
death by misfolding
cystic fibrosis
caused by a defect in a membrane bound protein called the cystic fibrosis transmembrane conductance regulator, the C, t, r.
12alpha helices span bilayer of surface
disease causing mutation deletion of Phe508 in CFTR which results in a misfolded protein that doesn’t insert into the plasma membrane
mucus in the lungs = breeding ground for microorganisms
death by misfolding: the Prion disease
misfolding degenerative brain disease Creutzfeldt-Jakob disease CJD in humans and bovine spngiform encephalopathy BSE in cosw and scrapie in sheep
the brain is riddles with holes
human PRPin monometic and dimeric forms
infectious agent 28kiladolton protein called prion protein infectious only
the misfolding of a prion molecules starts a domino effect resulting in more and more misfolded prion an leads to the disease state
the disease causing prion protein becomes a template for the transformation of the normal prion protein, which is in human brains and turns into a high beta sheet form of the protein which forms insoluble fibres in the brain,
which may well be an element of the toxicity to the nervous system with these infections.
death by misfolding alzheimers disease
Amyloid-beta Precursor Protein APP found on the surface of cells. throughout the body
its is membrane tethered and composed of multiple domains connected by flexible linkers
the intat aPP protein is a membrane recpetor that signals through G protein cascades inside the cells
it also binds extracellular matrix components outside the cell such as Heparin and Laminin
APP is cleaved by proteases called secretases (alpha, beta and gamma): the large extracellular piece controls nerve growth
the smaller intracellular piece interacts with the nucleus
Amyloid fibril formation by amloid-beta:
when beta and gamma secretase cut, the peptide is about 40 residues long but in resense of gamme secretase mutation associated with alzheimers it becomes 42 residues long
helical according to NMR in solvents in aq prone to form beta sheet structures in fibrous form
in disease form throusands of peptides stack ontop of each other - each interacting with its neighbours to form a long beta sheet
amloid fibrils form ense plaques on nerve cells which leas to the onset of demntia as the brain slowly oses normal function
so the samll amyloid beta peptide causes all the damage; as proteolytic fragment it leaves the membrane, changes shape and aggregates into long fibrils
unsuccessfully pharma try to; clear away using monoclonal antibodies that gain access to brain and we’ll clear away the fibrous deposits of the amyloid beta peptides
