Download Protein folding

Folding DNA and Proteins—WLC
When they have problems—Prion diseases
How proteins get help--Chaperones
Analysis: The Worm-Like Chain for elasticity
F= - kx
Hooke’s Law: You apply a force on something and it increases in length
linearly. Proportional constant = k. Minus sign because it’s a restoring force.
Force related to fractional increase (x/L). What if force isn’t proportional to
distance?
Need something more sophisticated: k is not a constant, independent of x;
As x gets bigger k gets bigger: F = f(k)x.
http://biocurious.com/2006/07/04/wormlike-chains
Two Models of DNA
(simple) Freely Jointed Chain (FJC)
& (more complicated) Worm-like Chain (WLC)
Idealized FJC:
Realistic Chain:
FJC: Head in one direction
for length b, then turn in
any direction for length b.
[b= Kuhn length = ½ P,
where P= Persistence
Length]
FJC: Completely straight, unstretchable. No thermal
fluctuations away from straight line are allowed
The polymer can only disorder at the joints between
segments
WLC: Have a correlation length
FJC: Can think of DNA as
a random walk in 3-D.
The Freely jointed Chain (FJC)
The molecule as a chain of perfectly rigid subunits of length b joined
by perfectly flexible hinges. Segment-to-segment angle = q
You can think of b as the length of the
repeating subunits, and is called the
Kuhn length (= 2 x Persistence length).
Persistence length: you start out going
in some direction: how long will you
tend to keep going: for DNA—about 50
nm, or 150bp.
If the molecule is under an applied force f as above, the effective energy for
the chain is given by
If there were no applied force, all configurations have equal energy (and
therefore the system has large configurational entropy), and the chain orients
itself in any which way—analogous to a random walk.
http://biocurious.com/2006/07/04/wormlike-chains
The Freely jointed Chain (FJC)
The equation can be solved and yields
From Science, 1992, Vol 258, pg.1122,
by SB Smith, L Finzi, C Bustamante
Direct mechanical measurements of the
elasticity of single DNA molecules by using
magnetic beads.
Force vs. Extension for DNA
F=-kx works well at very low force;
at higher force, DNA is extended (> 50%), need FJC or better is WLC
At very low (< 100 fN)
and at high forces (> 5 pN),
the FJC does a good job.
In between it has a problem.
There you have to use WJC.
You measure the Persistence length
Strick, J. Stat. Physics,1998.
WLC: A slight extension of FJC
A new phenomenological parameter has entered the energy term, A, which is
a measure of the persistence length of the chain, or how long a segment of the
chain will have tangent vectors all pointing in nearly the same direction.
Indeed, the tangent-tangent correlation function for the wormlike chain at zero
stretching force is given by
or, the similarity in directionality for the chain decays as an exponential in the
persistence length.
An analytic solution to WLC is not currently known, but the above equation
has been solved numerically.
At low force it again displays a Hookean linear relation, but as the extension
nears the contour length of the molecule, it scales not as 1/f as predicted by
freely jointed chains, but as 1/f1/2, in significantly better agreement with
the data.
The Worm-Like Chain (WLC) for elasticity
Increase in end-to-end length (x) of polymer:
Worm Like Chain (WLC) of entropic elasticity.
Force related to fractional increase (z/L)
where A = Lp: persistence length, a measure of the chains bending rigidity
= 2x Kuhn Length
L = contour length
z = extension
Each unfolding event increases the contour length of the homopolymer by a
constant value, ∆L.
WLC Fits very well at all stretches
Protein Mis-Folding & Chaperones
Protein Folding Summary
(From previous Protein Folding Lectures)
• Proteins can fold and do say fairly fast (< second).
• Protein Funnel is a good model. Extending beyong nearest
neighbor interaction: Molecular Dynamic Simulations
sometimes do a better job (with a lot of $$).
• ΔG is almost always small: (5-10 kT—few H-bonds). E goes
down; S goes down. They compensate.
• Kinetics – fast cause not huge barriers. (Detailed calculations
necessary.)
• In most cases, don’t need help. In complicated cases (big
proteins, very crowded conditions such as in a cell) proteins
get help: proteins called chaperones.
Protein folding: the energy landscape theory
Unfolded

Folded
Unfolded
ENTROPY

Active
IA
Molten
Globule
State
Intermedi
ENERGY
Inactive
Lattice Model: only worry about nearest
neighbor interactions. Hydrophilic and
hydrophobic interactions
Molecular Dynamics: write F=ma for everything
IB
Native sta
Misfolding of Proteins
Most proteins can spontaneously refold: Primary sequence determines tertiary.
Some proteins do not: boil an egg, bring temp back down and won’t re-form.
(Albumin goes from clear to milky white.)
Commonly the hydrophobic residues get exposed. When concentration of protein is
high, they can fold up with other proteins instead of with itself and remain unfolded
and aggregated.
Wide variety of proteins; similar structure, bad outcomes!
Amyloid fibers & plaques: Mad Cow diseases, Alzheimer
Disease, Parkinson Disease, maybe some forms of diabetes
Protein Folding in the Cell
•
•
•
•
Most proteins probably go through several stages on their way to a
stable structure. Some need help, particularly in crowded env of cell.
Chaperones are protein molecules that assist the proper folding of
other proteins. Often are heat shock proteins (HSP#) because they
help cell with elevated temperature which tend to cause proteins to
mis-fold. Also involved in newly synthesized proteins where a lot of
hydrophobic groups haven’t yet folded up properly.
Some chaperone systems (Chaperonins) work as foldases: they
support the folding of proteins in an ATP-dependent manner (for
example, the GroEL/GroES system). Other chaperones work as
holdases: they bind folding intermediates to prevent their
aggregation, no ATP required.
Diseases such as Alzheimer’s, Parkinson’s, and mad cow disease
are associated with misfolded proteins
• “Recent advances in single-molecule analysis have brought insights into
structural heterogeneity of chaperones, folding intermediates and affinity of
chaperones for unstructured and structured protein chains.” [Wikipedia]
When intermolecular contacts are significant,
need to modify energy funnel
Native contacts, %
Folding funnel
Stabilized by intramolecular
interactions
Unfolded
Aggregation funnel
Assume that 50% of
contacts are formed,
then can either fold
normally (i.e. in
dilute conditions), or
it can react with
another partially
unfolded protein and
fall down the
aggregation funnel.
ENERGY
Idealized case:
Stabilized by interchain interactions
Native
Aggregate
Partially folded state
Slightly More Realistic Scenario
(allow for formation of long fibrils)
Folding funnel
ENERGY
Nucleation
Polymerization
Aggregation
+
Fibril
Lysozyme: Well Studied example of mis-folding
S-S
S-S
W64R
N I56T
S-S
C
α-domain
D67H
β-domain
T70N
S-S
Lysozyme fiber
Lysozyme
form amyloid deposits in the gut
Lysozyme is abundant in a number of secretions, such as tears,
saliva, human milk, and mucus. Also in egg white. (Well studied.)
Damage bacterial cell walls through catalyzing. Forms amyloid fibers.
Simple, but excellent
Videos about Prions
http://www.youtube.com/watch?v=tfv3xAw0XOE
http://www.youtube.com/watch?v=pqhpVpafjmk
Prions– infectious agents that can be transferred
which are devoid of Nucleic Acids
Stanley Prusiner has added prions to the list of well known infectious
agents including bacteria, viruses, fungi and parasites. Prions exist
normally as innocuous cellular proteins, (generally called PrP). However,
prions possess an innate capacity to convert their structures into highly
stabile conformations that ultimately result in the formation of harmful
particles, the causative agents of several deadly brain diseases of the
dementia type in humans and animals. This is called PrPSC (where SC
standards for scrapie, the original prion-disease where it was discovered
in sheep). PrPSC is very stable, resistance to degradation by a number of
proteases, heating chemical denaturants…). Prion diseases may be
inherited, laterally transmitted, or occur spontaneously. Regions
within diseased brains have a characteristic porous and spongy
appearance, evidence of extensive nerve cell death, and affected
individuals exhibit neurological symptoms including impaired muscle
control, loss of mental acuity, memory loss and insomnia.
A prion in the Scrapie form (PrPSc) is an infectious agent composed of protein
in a mis-folded form. This is the central idea of the Prion Hypothesis. This
would be in contrast to all other known infectious agents, like viruses,
bacteria, fungi, or parasites—all of which must contain nucleic acids (either
DNA, RNA, or both). PrPsc can catalyze PrP to form amyloid fibrils.
Prions– Alzheimers and more (Nobel Prize 1992)
Prions are not considered living organisms but are misfolded protein molecules which may
propagate by transmitting a misfolded protein state. If a prion enters a healthy organism, it
induces existing, properly folded proteins to convert into the disease-associated, misfolded
prion form; the prion acts as a template to guide the misfolding of more proteins into prion
form. These newly formed prions can then go on to convert more proteins themselves; this
triggers a chain reaction that produces large amounts of the prion form. All known prions
induce the formation of an amyloid fold, in which the protein polymerises into an aggregate
consisting of tightly packed beta sheets.
http://en.wikipedia.org/wiki/Prion
The figure schematically illustrates how
variants of disease causing prions affect
different parts of the brain. In Bovine
Spongiform Encephalitis (cattle), the brain
stem is affected; in Fatal Familial Insomnia
(humans, a different mutation of the PrP
protein) the thalamus region; in CreutzfeldtJakob Disease (humans), the cerebral
cortex; in KURU (cannibalistic humans in
Papa New Guinea) and GerstmannSträussler-Scheinker (humans, inherited
form) disease the cerebellum is damaged.
http://www.nobelprize.org/nobel_prizes/medicine/laureates/1997/
press.html
Amyloid Fibers…involved in Alzheimers
Protein amyloid aggregation has been recognized as a major cause of several
important diseases, including Alzheimer’s disease (the fourth most common
cause of death in the Western world), Parkinson’s disease, type II or noninsulindependent diabetes, and the transmissible spongiform encephalopathies. About
17 different proteins have been found to form amyloid in vivo. Amyloid fibrils
formed from those proteins share some common morphological features, but
these proteins do not have a conserved sequence or native structural motif
Cao A, Hu D, Lai L. Formation of amyloid fibrils from fully
reduced hen egg white lysozyme. Protein Sci. 2004
Amyloid Fibers…involved in Alzheimers
Protein amyloid fibers are often found to
have a β-sheet structure regardless of their
sequence, leading some to believe that it is
the molecule's misfolding that leads to
aggregation.
There is a lower energy state which
is fibers—e.g. ameloid fibers–
multiple states!
http://www.informaworld.com/smpp/content~content=a7
79685983~db=medi~order=page
Enzymes act on the APP (Amyloid precursor protein) and cut it into fragments of protein, one
of which is called beta-amyloid and its crucial in the formation of senile plaques in Alzheimer
Chaperones—how proteins get help in folding
Figure 5.23
Chaperones
GroES/GroEL—(Class I) bacterial, mitochondrial,
1MD
In (Other
eukaryotes
the proteins
andnearly
Hsp10 are
and
class,
ClassHsp60
II, not
as structurally
well characterized)
functionally nearly identical to GroEL and GroES, respectively. Correctly
Polypeptide
folded
protein
Cap: GroES
Hollow
cylinder
GroEL
Chaperonin
(fully assembled)
Steps of Chaperonin
Action:
1 An unfolded polypeptide enters the
cylinder from
one end.
2 The cap attaches, causing 3 The cap comes
the cylinder to change
off, and the
shape in such a way that
properly folded
it creates a hydrophilic
protein is
environment for the
released.
folding of the polypeptide.
In general, poorly understood how works. Whether it is active in terms of
interactions between chaperonins, or inert, where chaperonines simply make a
environment conducive to folding, is not known. It is however, required for the
proper folding of many proteins.
Chaperones
(Bacterial GroES-GroEL)
With ATP, environmental conditions suitable for proper folding
ATP-dependent
folding along a
smooth energy
landscape
135Å
binding
[Don’t worry about details
but know via ATP hydrolysis,
that can get over the bumps
And slide down “easily”]
GroES
Misfolded protein
in kinetic trap
184Å
145Å
GroEL
Correctly folded protein
10º
ADP x 7
60º
20º
120º
GroES
ATP x 7
ATP x 7
ATP x 7
10s
45Å
ATP x 7
GroEL
Binding of a 7 ATP molecules to the GroEL ring triggers conformational
change that results in slight twist and tilt in the subunits and in exposure of
hydrophobic patches that interact with and help to unfold misfolded protein.
Class evaluation
1. What was the most interesting thing you learned in class today?
2. What are you confused about?
3. Related to today’s subject, what would you like to know more about?
4. Any helpful comments.
Answer, and turn in at the end of class.