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Protein Folding and Misfolding (Protein folding (Free-energy of folding…
Protein Folding and Misfolding
Protein folding
Anfinsen principle of protein folding
Protein can refold spontaneously by themselves
Conformation is determined by amino acid sequence alone
Anfinsen's Experiment (ribonuclease folding)
Secondary structure is broken apart by denaturant(urea), and the disulfide bonds are broken with mercaptoethanol
When urea and mercaptoethanol are removed, the ribonuclease refold by itself with correct folding
Two-state folding equilibrium
highly cooperative
rapidly interconvert between completely folded and completely unfolded (very transient intermediate)
localised “nucleation” of folding of nearby sequence
equilibrium constant
determined from rate constant K
eq
= k
F
/ k
U
determined from free energy ΔG = – RTlnKeq
at equilibrium, mixture of folded and unfolded state
Minoring folding/unfolding
CD - secondary structure
Fluorescence - whether it's buried
NMR
Free-energy of folding
monitor fraction unfolded at different [denaturant]
determine Keq at different [denaturant] (Keq=[Native]/[Unfolded])
calculate ΔG at different [denaturant]
#
extrapolate ΔG to [denaturant] =0 to bobtain ΔG(H2O)
although ΔG only slight negative, but protein generally entirely folded
Negative enthalpy from formation of bonds, positive entropy from hydrophobic collapse (release of structured water)
Folding kinetics
Measuring folding rate via stopped flow technology
Fast mixing between a solution with unfolded proteins and denaturant and a refolding buffer solution
Use detector (eg. CD) to measure folding
Another experiment with injection of folded protein into unfolding buffer (measuring unfolding rate)
Chevron Plot
logk vs. [denaturant]
extrapolating the folding curve to get kF, extrapolating the unfolding curve to get kU.
If the folding/unfolding curve is not linear, the folding can involve more than 2 states.
via fluorescence dye
mixing a protein with a hydrophobic fluorescence dye
When heated, the protein starts to unfold, dye binds to the exposed hydrophobic patches, brighter in fluorescence
After certain temperature, protein begins to aggregate and the fluorescence decrease
Application: VSVG protein mutant
Temperature sensitive mutations:
less negative 𝚫G(H20),
more sensitive to temperature change
unfolded at 40 °C, retained in ER; refold at 32°C, secrete into Golgi
Use VSVG to study the mechanism of vesicular trafficking from ER to Golgi
Folding Trajectory
Transition state
undetectable
Adopt higher energy state than unfolded protein
(energy barrier)
Φ analysis
the importance of particular chemical moiety
in a protein structure on
stabilizing a transition state
use site-directed mutagenesis
conservative mutation
only remove interaction (not introduce new interaction)
should not remove charges (affect other interaction)
choose conserved/buried residues
Φ = 𝚫𝚫G(‡–U) / 𝚫𝚫G(F–U)
use Chevron plot to get folding rate from mutation(kF') (𝚫𝚫G‡–U= -RTln(kF/kF’)
If mutation does not change the folding rate, then Φ =0
If both kF and kU are changed, 0<Φ<1
Φ =1
the moiety is important to the stability of transition state,
lies at a structured site in the transition state
0<Φ<1
additional processes are occurring (eg. mixture of transition states (different trajectories), conformational reorganization at the mutation site)
If two proteins share the same fold, but ) analysis marks different residues as being structured in transition state, two protein must fold through different transition state (trajectories)
Models of folding
Nucleation Condensation
Secondary and tertiary structures form together at the
nucleus
Then remaining structure forms around nucleus
Example: Chymotrypsin inhibitor 2 (CI2), helix and sheet have structure in transition state
Hydrophobic collapse
Residue collapse together (hydrophobic effect)
Then secondary and tertiary structure form
Framework
Secondary structure form first
Then tertiary contacts form
Protein Dynamics Stimulation
for protein with known structure
Define parameter (eg. temperature, volume, density)
Initiate unfolding by increasing temperature
Motion of atom based on their own kinetic energy and forces exerted by other atom
Advantageous
: Folding trajectories can be viewed precisely by simulation
In the example of Cl2
#
Good agreement between simulation (S value) and experimental Φ analysis
both S and Φ value predict transition state comprises of residue local and distal in sequence.Hence, nucleation condensation.
#
Folding landscapes
Multiple-state folding
the plot of Fractional unfolded vs. [denaturant] consists of multiple sigmoidal curve
stable intermediate accumulate in the folding pathway
Example: Apolipoprotein E
#
three isoform: apoE2, apoE3 and apoE4. ApoE4 can lead up increase of risk in Alzheimer's.
only one amino acid difference in sequence, but large difference in the folding properties
ApoE4 poorly fit to the 2-state model
Experiment: mixing ApoE with pepsin protease and different concentration of urea
when the sequence unfold, it will be cut by proteases
some regions is more resistant to protease (more structured in intermediate)
use mass spectrometry to identify the cut sites
from the cut sites, a "
molten globule
" intermediate model can be built up
protein retains near-native secondary structure
hydrophobic core not packed
conformation overall sightly expanded and "molten"
Levinthal Paradox
protein folding cannot occur by random sampling of all conformation
protein folding is sped up and guided by local interaction.
Local amino sequences which form stable interactions can serve as nucleation points in the folding process
Lattice model
consider protein as beads connected by one string
bead interact with each other by pairwise contact potentials
each additional contact lower the energy (ie. unfolded has the highest energy, the native state has the lowest energy)
Q0=native contact; C=total contact (Q0=C at folded-state)
small random changes are made and the conformation with lower energy is favoured
plotting ad 3D diagram for all conformation gives a
funnel shape
(Q0 vs. C vs. F(free energy))
unfolded state: Q0=0, C=low, F=high
intermediate: Q0=medium, C=high, F=lower
folded state: Q0=highest, C=high, F=very low
folding funnel (energy relationships with structure)
different protein has different folding landscape.
2-state model: smooth landscape; multiple-state model: rugged landscape (energetic barrier)
intermediate accumulate when trapped in local minimum
takes more energy (hence more time) to bump out of the minimum
example: molten globule
#
change in environment can alter the landscape
Example: folding pathway of hen egg white lysozyme
folding is monitored by "stopped flow" kinetics, and the contacts are tracked using
hydrogen/deuterium exhcange
stopped flow experiment
diluting protein into D2O + buffer to initiate folding
at different time point, diluting into H2O + buffer for H/D back-exchange
at t=0, entire protein is still unfolded back-exchanges to H (low mass)
at t=0.1s, some positions are protecting, the intermediate mass suggests intermediate formation
at t=2s, protein is folded and protected from back-exchange (high mass) (surface residue?)
quench the exchange by dropping pH to 2.5
protein is allowed to completely fold and is analysed via NMR or Mass spectrometry
change in mass indicates the extent of H/D exchange
Mass spectrometry
#
NMR analysis
Deuterium is invisible by HSQC
the intensity of each amide resonance is proportional to the extent of H back-exchange
enable to look at each residue
protein is deuterated and kept unfolded in D2O + denaturant
experimental evidence suggests multiple folding pathway
hydrogen/deuterium exchange
hydrogen in water and aminde group can exchange
Side-chain hydrogens exchange too fast to detect
hydrogens do not exchange whilst forming a hydrogen bond
stable secondary structure has many hydrogen bonds
unfolded sequences has little hydrogen-bonded amide hydrogens
exchange rate is fast at pH7 (faster than protein folding), but slow at low pH (H/D exchange can be quenched by dropping pH)
using stopped-flow kinetics
addition of deuterated water to unfolded protein to initiate folding and exchange (?)
quench the exchange by dropping pH to 2.5
Misfolding
Rival folding funnels
when protein flips from a normal folding trajectory into a non-normal one via intermediate conformation
change in environment can change the landscape and favour the intermediate
increase the risk of triggering non-native folding options
accumulation of intermediate confomation allows more non-native contacts to form
in the abnormal funnel, aggregation offers a new route to reach lower energy states which compete with the proper folding pathway
factors that can promote aggregation pathway
mutation
environmental changes
post-translational modification
changes in ligand interaction
Amyloid
generic structural motif characterized by a fibrous morphology
β-sheet core structure
formation is nucleated (cooperatively) and only slowly reversible
very low energy state that is comparable to native state
end product in many neurodegenerative diseases
fibrillar structure can be observed using negative staining TEM
additional electron dense dye coating
only observing the shadow of the fibrils, hence inherently low resolution
can be improved using CryoTEM
combining low contrast individual images
able to define structural characteristics of the fibrils at high resolution
Three examples of protein misfolding
ApoE
Fraction of unfold at different [urea]
ApoE4 is more sensitive to unfolding (low ΔG)
Urea (denaturant) changes the energy landscape to favour unfolded forms
Gel Filtration
gel filtration sorts molecules based on their size (
larger
molecules elute
earlier
)
ApoE4 forms aggregates more rapidly (more elute at earlier time)
Negatively stained TEM
#
aggregates of apoE appear as small twisted amyloid fibrils
equilibria affects aggregation
Native state: ApoE2>ApoE3>ApoE4
mistfolding pathway: ApoE4>ApoE3>ApoE2 (accumulation of partially unfolded state favours amyloid pathway)
Huntingtin
Pelletability indicates aggregation
long polygulatamine repeat lengths induce huntingtin to aggregate
aggregates are pelleted by high-speed centrifugation
aggregates have different tertiary/quaternary structure
react differently to different conformational specific antibodies
forming different morphologies (TEM)
expansion of polyQ enables new misfolded states to be populated as well as diverse aggregation pathways
immuno-staining
in normal brain cell, huntingtin is diffusely distributed
in disease brain cell, huntingtin forms dense agglomerates within cell (inclusions)
Tau
microtubule-binding protein that forms aggregates in Alzheimer's and frontotemporal dementia
normal function: stabilize microtubules
the binding of Tau to microtubules is regulated by phosporylation
when dephosphorylated, the Tau binds to microtubule; when phosphorylated, it detaches
expressed as 6 isoform, result from differential splicing
mutation influences Tau aggregation
phosphorylation
detached tau can undergo aggregation pathways
Hyper-phosphorylation increases the concentration of detached tau
#
affect microtubule binding (affinity)
aggregation rate
gene splicing
#
mutation can change folding landscape, and enable new aggregation pathways
folding in a crowded cell
excluded volume effects
molecules in a solution occupy space
packing of the molecules can change their interaction with each other and their conformation and folding
excluded volume affects different molecules differently
small molecules can fit in the gaps between large molecules
crowding is more acute for large molecules in confined spaces
large molecular requires more energy to fit in crowded space
energy-molecule relationships: μ(ex)
varies with size, shape , crowdedness
protein concentration + surrounding crowdedness
effect of excluded volume
improved self-association behaviour
trimer takes up less volume than monomer + dimer
crowding increases μ(ex) less for trimer
change the energy level (free energy= initial free energy + μ(ex)) and favour polymer formation
Example: apoC-II
crowding increase the rate of amyloid formation
spontaneously form amyloid in neutral buffers
use glucose polymer "dextran" to mimic the crowded cell environment (higher [dextran], more crowded)
retarded diffusion
altered kinetic rates and equilibrium condition
promotion of ordered packing
Compartmentalization
Each compartment has a cloud of specialized machinery to collectively work together and help keep the protein folded
Endoplasmic reticulum
govern the quality control of proteins and remove/repair not properly folded protein
Calnexin cycles
use chaperones and glycosylation tag to fold complex protein
N-linked glycosylation (N:asparagine)
glycosylation pattern "tag" the foldedness of the protein and confer other function (reprocess/degraded) to the protein
Calnexin
a chaperone which binds to a protein and help it fold
if successfully folded, distinct glycosylation pattern signal the cycle to deliver the protein to the cell
if not properly folded, different glycosylation pattern indicates it need to go through the cycle again (or be further processed)
ERAD
after quality control from calnexin cycle, proteins that are deemed unfolded are expelled from ER and be degraded
a certain glycosylation tags trigger export to the cytosol for degradation
Pulse Chase
detect the longitudinal fate of molecules using transient labelling
pulse the cell: add radioactive amino acid ([35S] methionine) for a short time (all new protein produced during the period are labelled)
chase the cell: harvest cell at different time interval and capture proteins of interest by immunoprecipitation
radioactive amino acid is wash out
run proteins on a gel and scan the gel for radioactive protein (protein with glycosylation tag has a higher molecular weight)
Chaperones
function
assist protein folding correctly by minimizing incorrect folding pathway
prevent nascent chains from aggregating
HSP chaperones
Trigger factor (TF) make initial contact with nascent peptide being produced to prevent aggregation
HSP40 and 70 recognize and hold hydrophobic and unstructured amino acid sequences (allow native fold to form)
HSP40 binds to the protein and recruit HSP70 which clamps on the peptide (ATP-dependent step)
the hydrophobic-rich residues of the unfolded protein binds to the β-sheet of the HSP70 peptide binding domain and the α-helix clamps on
HSP70 has high affinity to the peptide
nucleotide exchange factor release ADP and return HSP70 to low affinity state and peptide is release
each cycle allows protein to
incrementally fold
different domain can form separately (
folding of large complicated protein
)
sometimes deliver protein to chaperonins for folding
Chaperonins
a closed chamber to assist individual proteins to fold; changes the energy landscape to favour folded forms
limited to small proteins (<60 kDa)
TRiC in mammal
CryoEM shows that when ATP-bound it's closed conformation; when ATP-free, it's open conformation
GroEL/ES in bateria
once the unfolded peptide enters, the chamber closes (ATP dependent process)
the unfolded proteins binds near the lid via a hydrophobic patch
when the lid close, the chamber becomes hydrophilic
it forces the protein detach and hydrophobic contacts to form inside the protein
the protein allowed to fold inside the chamber (disfavours non-native folding pathway)
hypothesis 1: promote reaction from the intermediate state and help break non-native interaction
hypothesis 2: remove the possibility of intermediate state
vectorial folding
protein tethered to ribosome can restrict available conformations in the folding funnel (geometric constraints) (biases away from abnormal folding pathway)
Example: Green fluorescent protein (GFP)
#
β-barrel with fluorophore in the barrel core; only form fluorophore when fully folded
folding is not reversible when denatured
stalling sequences are inserted at the end of the GFP sequence, causing ribosome to jam the leaving GFP peptide
two stall position are created
one enable the whole GFP comes out of the exit tunnel
one traps part of the last β-strand in the exit tunnel
GFP fluorescence yielded indicates the extent of folding
Experiment shows GFP cannot fully fold until the c-terminus is comepletely released (but still not at maximum efficiency)
experiment also show that folding is more efficiently when tethered into ribosome than refolding from solution
Frontiers of folding
basic of fluorescence
the emission wavelength has lower energy than the absorption wavelength
fluorescent protein
entirely genetically encoded, can express in cell and tag to protein as a marker
fluorophore involves an autocatalyzed cyclization reaction of three residue in the middle of the β-barrel
FRET: fluorescence Resonance Energy Transfer
fluorescence-based strategy to monitor orientation and distances
two fluorophores within a few
nm
can transfer energy to each other (acceptor absorbs some of the energy non-radiatively from the donor and emit its fluorescence)
FRET depends on the distances and orientation
basic parameters
FRET efficiency E(FRET) (decrease significantly after certain distance)
Distance where E(FRET)=50% (R0)
proportional to: orientation factor, quantum yield (efficiency of the fluorescence when shine light on it), spectral overlap (the overlap of the donor emission and acceptor absorption spectrum)
inverse proportional: refractive index of solution
orientation factor κ2
fluorophore is planar and absorb light in orientation-dependent manner
usually assume dynamic freedom approximates the sidechain behavior (κ2 = 2/3)
Example 1: Tau protein conformational changes upon phosphorylation
Tau is mutated to contain one fluorescence donor and one acceptor
donor: tryptophan
acceptor: IAEDANS (attached to Cys)
different mutant with Trp and Cys at different locations
additional mutation to mimic phosphorylated form of tau
FRET efficiency is different for different mutation pair, which infer the distance between two residue and enable mapping of conformational changes
FRET can also track unfolding
FRET changes at different [denaturant]
the FRET pairs report on localized changes in as tau unfolds
higher [denaturant], higher donor's fluorescence, less acceptor fluorescence
Example 2: orientation of fluorophores on DNA
donor fluorophore at 5' end; acceptor fluorophore at the 5' end of complementary strand
Since DNA is relatively rigid and periodic, can use this property to study κ2
as DNA shorten, the orientation of the fluorophore will twist
stimulation assuming completely rigid shows periodicity of peaks and as distance increase, the peak is lower
experimental data show the DNA is not completely rigid, there is some mobility due to the flexible linker. Also, the dynamic average (κ2=2/3) assumption is acceptable
Single molecule folding
bulk phase: average signal of an ensemble of molecules
lose of information encoded in single molecules
Trajectory of folding
variation of structure
single molecule FRET
use highly sensitive detector to measure photons emitted from a single fluorophore
using dilute sample to isolate single molecules
Approach 1: confocal fluorescence (watch molecules diffuse in and out of the volume)
Approach 2: traaping single molecules on a surface (either entrapment in phospholipid bilayer vesicle or tether to antibodies)
Example 1: folding of Chymotrypsin Inhibitor 2 (CI2)
label CI2 (wild type and mutant) with a FRET pair
confocal imaging of dilute solution
solution has an equilibrium of folded and unfolded protein
unfolded protein will show a large donor fluorescence and a small acceptor fluorescence
each molecule has its FRET efficiency
Measure FRET at different [denaturant]
create a histogram of all FRET efficiencies
E(FRET) fell into two Gaussian population, indicating 2-state unfolding
mutant completely unfolds at lower [denaturant]
mutation K17 destabilizes the conformation
Example 2: clathrin coat disassembly
clathrin forms coats to drive endocytosis.
auxilin helps to buildup the clathrin coats
HSC70 interacts with auxilin to break apart the cage unit
once the clathrin coat buds off vesicles, the cage falls apart into unit
label clathrin with one fluorophore and label Hsc70 with a different coloured fluorophore
clathrin coats are fixed to a slide with antibodies and each coat is spaced far apart
through fluorescence imaging, we can observe the clathrin recruits Hsc70 and the Hsc70 breaks down the clathrin (disapparence of fluorescence)
therapeutic strategies
Proteostasis
active mechanism (integral machinery) to maintain folded proteins and remove misfolded/aggregate protein
#
proteostasis can target any stage of the folding/misfolding pathway
disaggregation chaperons can suppress/reverse the misfolding pathway
folding chaperones can minimise the accumulation of intermediate state which is vulnerable to misfolding
Chaperones responds to aggregating protein
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chaperones are up-regulated
Example: Ataxin-3
long polyQ mutation cause neurodegenerative disease
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different distribution pattern for different Q-length (long polyQ length forms aggregates and shows as spots)
HSP70 accumulate at the site of aggregated ataxin-3 (
HSP70 engages with aggregated protein
)
over-expressing HSP70 can protect flies from eye tissue degeneration by ataxin-3 (Q78) (
extra HSP70 can help restore proteostasis
)
overstraining proteostasis
Example: Par(ts)
Par(ts) is a temperature sensitive protein
#
at 15 °C, it is a smooth distribution (no aggregation); aggregates at 25 °C
when poly-Q protein is introduced, par(ts) starts aggregate at 15 °C (not at the same site as poly-Q protein)
there is only a finite reserve of resources, the introduction of misfolding proteins can divert the resources, and insufficient resources to prevent Par(ts) from aggregating (also other protein can become unstable)
the intrinsic folding equilibrium can not be changed, but can smooth the folding funnel (reduce the accumulation of intermediate state)
Therapeutics for misfolding disease
Case 1: Cystic fibrosis
Caused by mutation in CFTR protein which leads to misfolded CFTR (folding of CFTR is kinetically driven- undergo a series of incremental step to fold)
mutant is efficiently removed by the quality control system
folding is disrupted at co-translational and post-translational steps
cotranslational folding on ER membrane (vectorial folding)
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ΔF mutation in the NBD1 domain destabilizes other domains
post-translational folding in Golgi where domains assemble to form final structure
in wild-type: a highly packed, low free energy protein forms
in mutant: domains can't assemble efficiently, retain non-compact structure (molten globule like)
#
proteostasis degrades the CFTR
Freshly made CFTR in ER: when calnexin fails to fold CFTR, it is directed to ERAD for degradation
Mature CFTR on plasma membrane: targeted by quality control, through endocytosis, it gets rescued by QC or degraded in lysosome
Strategies to rescue CFTR recovery
drug 1: binding to the NBD and stabilise the native fold
drug2: reducing detection of mutant CFTR by QC
assess efficiency of folding
express ΔF508 CFTR that normally can't transport halide
express YFP (yellow fluorescence protein, quenches by iodide) in the cell
add iodide to the cell culture
faster quenching rate = more functional CFTR
drug were screened that improved halide transport
Case 2: transthyretin
mutant transthyretin (TTR) causes familial amyloid polyneuropathy, characterised by massive accumulation of TTR amyloid fibrils
aggregation proceeds through misfolded monomer (dissociation of tetramer to native monomer, then partial denature to amyloidogenic monomer)
V30M reduces the energy barrier of transition state to monomer
monomer is vulnerable to rival misfolding pathway but not energetic favourable under normal condition
suppressor
of the amyloid disease (T119M mutation)
express V30M TTR and T119M TTR with acidic tag (FT2)
mix and form chimeric tetramers (5 possible forms) which then are separated using charge difference
Assay different tetramer for their stability and amyloid fibril formation rate
T119M conferred stability of tetramer (increase the energy barrier of the transition state)
T119M suppressed fibril formation
use
drug
to stabilise tetramer
TTR tetramer has two thyroxin binding site, thyroxine binds weakly but can stabilise the oligomer and mediate the dissociation
require a high affinity mimetics of thyroxine
a library of thryoxine
analogues
with
no biological hormone function
is made and tested for their influence on tetramer stability and amyloid formation
drug has variable effects on dissociation and fibril formation, but there is a good correlation between drugs that inhibit dissociation and those inhibit fibril formation
binding of a ligand lock TTR into lower energy state far away from misfolding pathway, hence suppress aggregation