Download Protein Folding

FUNDAMENTALS 1: 11-12
FRIDAY, AUGUST 13TH, 2010
PROFESSOR
I.
II.
III.
IV.
V.
VI.
VII.
PROTEIN FOLDING
Scribe: CHRISTINE SIRNA
Proof: THI TRAN
Page 1 of 4
PROTEIN FOLDING[S1]:
a) No definitive way to explain why every protein folds the way it does
b) How do you go from 1D primary structure to a triple helical or globular structure of magnitudes much larger
than you would have with a single subunit and how do you get to that particular point with fibrous or globular
proteins? No definitive answers but many considerations
c) Folding of proteins is a major problem in modern health systems
RNAase[S2]
a) First material studied with protein study was ribonuclease
b) small protein with 4 disulfide bonds
c) In picture the disulfide bonds are blue, yellow, green and pink
d) Number of AA is 124 from Amino terminus to C terminus
RIBONUCLEASE EXPERIMENTS [S3]
a) Chris Anfensen at NIH decided to study proteins and how they folded and they put them in Bmercaptoethanol and 8M urea
b) 8M urea is a chaotrophic solvent (promotes disorder) that denatures proteins
c) This is an example of denaturation, it is unraveled and all structure is lost
RESORATION OF ACTIVITY [S4]
a) How do we renature? If they allowed the protein to be oxidized (they reduced it to denature it)
b) Allowed it to be oxidized to a certain extent and added B mercaptoethanol to help rearrange disulfide bonds
and in a matter of seconds original structure was reestablished
c) Mathematically, you would expect the fully denatured protein to renature perfectly only 1% of the time but
they got 100% activity back
CONCLUSIONS TO RNAase STUDIES[S5]
a) Concluded that the native conformation of a protein is the state with the lowest Gibbs Free Energy
b) Everything that is spontaneous goes to a low free energy state
i. If it goes to a state where there is no more energy to be lost and it is satisfied and it is stable then it will be
preserved unless something else happens
c) Proteins follow unique paths to attain native state
d) Primary structure possesses sufficient information for proper folding
e) Most of these conclusions have been remodified over the years
LEVINTAL’S PARADOX [S6]
a) Levinthol came back a few years later reiterated some of Anfensen’s conclusions and said
i. if you take a small protein with 100 AA that can assume 3 positions (if a protein is denatured it can assume
100 different positions not a fixed protein and side chains can rotate).
ii. Then total possible structures 3100 = 5 x 1047
b) If you examine each structure for 1 x 10-13s
c) Then total search requires 5 x 1034s
d) This is 1.6 x 1027 years, a period longer than the age of the universe!
e) So therefore proteins don’t go through every gyration possible. They follow a pathway to get to the native
state.
ENERGETICS OF FOLDING [S7]
a) In thermodynamics you have the Gibbs Free energy equation
b) ΔG = ΔH – TΔS
c) Where: ΔG = free energy, energy available for work
i. if the sign of G is (-) the reaction has given off energy and does not require energy to proceed and is
spontaneous
ii. if it is a positive then the reaction only went off because we added energy.
iii. Ideal thing for chemical reaction especially one dealing with biochemistry is to have delta G be (-) because
you want something that provides energy.
d) ΔH = enthalpy, a measure of bond energy
i. bond formation (-)
ii. measure of bond breaking (+)
e) T = temperature, K
f) ΔS = entropy,
i. a measure of increasing chaos (+), chaos develops
ii. a measure of decreasing chaos (-), order is being established.
g) Natural thing is to have disorder being created
FUNDAMENTALS 1: 11-12
Scribe: CHRISTINE SIRNA
FRIDAY, AUGUST 13TH, 2010
Proof: THI TRAN
PROFESSOR
PROTEIN FOLDING
Page 2 of 4
h) Want ΔH to be negative and ΔS to be positive so that chaos and disorder is forming and this makes reaction
go nicely
i. Ex. If you cleave a polypeptide chain into all of its amino acids. That is increasing disorder from one nice
polypeptide chain to 2000 or so amino acids. This increases positive ΔS
ii. Want ΔG= +ΔH-TΔS and this would give you a --ΔG. This would be an optimal rxn.
iii. For a spontaneous process, ΔG must be (-)
iv. Is the folding of a protein a spontaneous process? YES. It does occur so you would expect ΔG to be minus
VIII. FOLDING OF GLOBULAR PROTEINS [S8]
a) ΔG is (-), bond formation and/or an increase in disorder must predominate
b) To go from denatured protein to folded protein or to go from random coil to a nice organized globular protein
like hemoglobin subunit you have to break some bonds with the environment.
i. Bonds must be broken from polypeptide chain and all bonds with water must be broken.
ii. New bonds must form as protein folds in on itself.
c) When a protein folds on itself (globular protein) there isn’t a lot of space, the atoms occupy about 80-85% of
the volume.
d) The change in ΔH is minimal. If anything it might be a little positive might be a little negative. No real
supremacy of forming or breaking of bonds.
e) Thus, ΔS is the deciding factor. Basically you increase this order b/c all of the hydrophobic side chains in this
polypeptide chain were organizing water. When they leave the water and go to the globular protein, water
can be disorganized. It leaves the clathrate structures and you have the situation where ΔS is a positive
because all the water molecules held together by the unfolded protein can roam around the folded protein.
f) This type of folding is ENTROPY driven. Driven because entropy is increasing in the medium in which the
protein is folding. This is why the proteins fold spontaneously.
IX. DATA SUPPORTING ENTROPY AS A DRIVING FORCE FOR FOLDING [S9]
a) Heat Capacity upon denaturation of protein increases because water is more highly structured
b) Takes more calories to cause the molecules of water to bounce around when highly structured then when
the water is free to move around by itself.
c) On refolding, heat capacity of water decreases because you have lost all the structure of the water that was
available here and now the water molecules are free to move. I takes less energy to cause the water
molecules to move and heat up once the protein has refolded
d) Also, if you take folded protein and add alcohol to the solution you decrease the differential between the
inside of the protein, which is very hydrophobic, and the outside, which is hydrophilic because you add
alcohol.
i. You make the water less hydrophilic and the protein differential is not so great. So protein can begin to
unfold. There is no driving force for protein to go and hide if the environment is becoming more
hydrophobic on the outside. So protein will start to unravel.
X. FOLDING OF FIBROUS PROTEINS [S10]
a) With a fibrous protein such as collagen or myosin or fibrin, there is no collapse or folding back on itself like
there is in a globular protein.
b) So those proteins essentially are not escaping water and in fact the primary structure of those proteins are
such that there is no tendency at all to avoid water. Love aqueous environment.
c) They have a lot of side chains that are polar.
d) When the chains come together to fold and wrap around each other, the formation of tertiary structure is
much different. New bonds are formed and the folding of these proteins is ENTHALPY DRIVEN.
i. This is a matter of making new bonds, new hydrogen bonds, and new hydrophobic bonds which cause
those particular molecules to fold
ii. This comes back to the dichotomy between fibrous and globular proteins.
XI. BOTTOM LINE ON FOLDING [S11]
a) When people talk about folding of protein think about whether it is fibrous of globular first!!!!
b) Most important message!
i. ΔG on folding of a protein is really -5 to -15 kcal/mole
ii. In comparison, If you have a mole of methane gas and add a spark you will have ΔG of about 5000
kcal/mole
iii. Protein folding is a low energy producing system, not a very robust system. Proteins do not fold rapidly and
with great energy and do not become quite stable.
c) Low energy involved in protein folding tells you the evolution of proteins has favored flexibility
d) Native proteins are on the borderline of denaturation
i. Most collagen molecules that do not get incorporated into fibers simply unfold b/c of low energy of folding
e) Misfolding is a common occurrence
FUNDAMENTALS 1: 11-12
Scribe: CHRISTINE SIRNA
FRIDAY, AUGUST 13TH, 2010
Proof: THI TRAN
PROFESSOR
PROTEIN FOLDING
Page 3 of 4
f) Only about ½ proteins synthesized actually get used, others are degraded in proteosome
g. Flexibility: hemoglobin needs it to move around and be functional. Therefore rigidity is not favored.
XII. PATHWAYS OF FOLDING [S12]
a) Molten globule: that tertiary structure or globular structure which almost is there but the last little bit of
structure needs to be formed.
b) Can tell when a system is in this state v. final state by molecular via chromatography
c) As protein continues its folding process it becomes smaller and smaller until it levels off to a final globular
state
d) In this picture it is still loosely structured and the final structure requires some alteration of the molten
globular state.
XIII. MOLECULAR CHAPERONES [S13]
a) Need chaperones!! This was totally not thought of in Chris Anfensen’s time.
b) Variety of proteins when being synthesized and finally folding need some help and this is done by structure
called groell structure (in bacteria). We have another system that is analogous to the groell structure called
TCP1 structures.
c) As a protein is synthesized on the ribosome, there is a danger that it will begin to complex and form
aggregates with new protein coming from a neighboring ribosome, so proteins need to be protected in that
area. This is done by the hsp70 system.
XIV.
PROTEIN FOLDING PATHWAYS [S14]
a) HSP= heat shock protein because the chaperones were first discovered as proteins which are made in
abundance when a cell was shocked by heat or some other noxious agent.
i. Protect newly synthesizing protein while being synthesized.
ii. When hsp70 proteins are released, if protein still needs to be folded properly it can go into chambers where
it can be folded in private.
b) Folding is taken care of very diligently by proteins in cell called foldases.
XV. STRUCTURE AND FUNCTION OF THE GroEL-GroES complex
a) Picture of cell cylinder in which protein folding takes place
i. unfolded protein enters in and is seen by the yellow parts of the cylinder (yellow parts are the really
hydrophobic agents that enable protein to be completely unfolded so it can fold again)
ii. ATP comes in and the configuration of cylinder changes and protein is allowed to refold properly
unencumbered by sides of cylinder
iii. After this, in a matter of seconds ATP is converted to ADP and new ATP molecules are added to the
bottom part of the cylinder. The cylinder opens up and the fully formed protein can be released.
b) This is a chamber, similar to proteosome chamber, is composed of many subunits of amino acids. This is
the beginning of life or birthing chamber and the proteosome chamber is the end of life of the protein. Both
are more or less cylindrical arrangements.
XVI.
ADDITIONAL FACILITATORS
a) Facilitators like protein disulfide isomerase (enzymes that cause enzyme to be unraveled, reduce disulfide
bonds and cause protein to start over again and refold).
b) Peptidyl prolyl isomerase; proteins that have proline are prone to have cis peptide bonds
i. Proline is unusual AA and can rotate different ways than most other AA
ii. Protein has to be isomerized back to trans peptide bonds as opposed to cis. This is done by peptidyl prolyl
isomerase
XVII. PICTURE
a) Ken Dill has proposed a funnel to show how high energy proteins are unfolded and as they get more folded
they go down in energy scale
b) At lowest energy level it is fully folded
c) Energy given off contributes to the minus ΔG
XVIII. HUMAN BIOCHEMISTRY
a) Disease of protein folding
b) Problems arise when proteins fold wrong.
1. Alzheimer’s:  amyloid peptide somehow gets cleaved from major protein in surface of cell and goes
into brain tissues and precipitates as mass of protein. They think this causes Alzheimers
2. Familial amyloidotic polyneuopathy: caused by transthyretin (previously called prealbumin), which
starts to precipitate in all kinds of tissues. One becomes logged with transthyretin.
a. Transthyretin is the chief amyloidotic protein but Alzheimer’s is also an example.
3. Protein p53 which when improperly folded because of a mutation is no longer able to inhibit
apoptosis and allows cell to grow and become metastatic cancerous cells
FUNDAMENTALS 1: 11-12
Scribe: CHRISTINE SIRNA
FRIDAY, AUGUST 13TH, 2010
Proof: THI TRAN
PROFESSOR
PROTEIN FOLDING
Page 4 of 4
4. Creatzfelt- Jacob disease: human equivalent of mad cow disease caused by prion proteins. Prion
proteins are proteins that have become synthesized by nerve cells and have become proteins of the
nerve cell membrane and then they change configuration from a protein with lots -helices to one
with -pleated sheets.
5. Hereditary emphysema: alpha1-antitrypsin is mutated and it cannot be secreted from the liver cells
where it is made. Alpha1-antitrypsin’s major job is to stop the activity of elastase. When elastase is
uninhibited it dissolves elastin (protein of lung cells that allows us to exhale without any energy). You
can inhale but exhaling is a matter of lung contraction. Elastin of the lung tissue contracts once
inhalation has occurred. If don’t have elastin and elastase eats away at elastin of the lung tissue, you
get emphysema (occurs as a result of the alpha1-antitrypsin to be properly folded).
6. Cystic Fibrosis: problem with mucous membrane of airways filling up and cutting off ability to
breathe. Result of mutation in protein that is supposed to function in trachea and upper part of the
lungs.
XIX.
GENERATION OF ALZHEIMER PEPTIDE
a) Amyloid precursor protein is composed of smaller protein coming from a large protein and depends on how it
is derived.
i. -secretase and a gamma secretase that operates on this particular protein.
ii. Dark yellow line in picture is amyloid protein that traverses nerve cell membrane and goes into the cytosol
of the membrane.
iii. If -secretase cleaves at this point, and kicks off remainder of polypeptide, there is no problem. Gamma
secretase can come along and cleave down in this region and the peptide that is released, the p3 peptide,
there is no problem
iv. However, If -secretase cleaves right here and gamma cleaves where it normally does down as residue 42,
then that particular peptide that is released is an amyloid beta protein (the one that causes the protein).
v. Depends on cleavage point as to whether amyloid plaques form in the brain
vi. Some people have amyloid plaques and are normal and some have Alzheimer’s disease with no plaque
but the above explanation is the general thought.
XX.
PRION PRECURSOR PROTEIN
a) Protein anchored in the membrane on nerve cells and it does not traverse membrane of nerve cells. Has a
large segment protruding outside cell. It composed largely of alpha components and has disulfide bonds
from one alpha helix to another and has carbohydrates attached to Asparagine residues in the alpha helices.
XXI.
TRANSFORMATION OF PRION PROTEIN
a) When the protein is liberated from cell, maintains alpha helix for a while, then denature, and renatures as a
protein, which has totally -pleated sheet.
b) -pleated sheet protein can influence further molecules and form large aggregates of proteins with -pleated
sheets that precipitate in nerve cell. Found in mad cow disease and Creatzfelt-Jacob (humans) disease.
c) No cure
XXII. PRION VARIABILITY
a) Various strains
i. Can be faulty formed prion protein can have influence on other protein and can cause it to take that shape
and gives you a polymerized protein of a disease a with certain manifestations.
ii. Prion protein can have slightly different shape, which will cause its neighbors to take on that different shape
and give you other form of the disease.
iii. There are several forms of the prion disease situation.
NO slide for alpha1 antitrypsin but
-the problem is derived from the fact that liver cells cannot secrete alpha1-antitrypsin that is not properly folded
and the liver tissue retains the alpha1-antitrypsin.
-you get lung problems with its absence as well as liver disease because of protein buildup.
[End 37:50 mins]