I.



II.
Double stranded Base-paired Helical DNA introduction
B-DNA -> obtained in vivo when DNA is felly hydrated.
A-DNA -> Dehydrated conditions (lab)
Z-DNA -> Left handed DNA
B-DNA structural Features.







B-DNA Diameter: 20 angstroms. (how many angstroms)
o Right-handed helical “staircase”
o 10 bases per Turn , so a base every 36° (degrees?)
o 3.4 A is the spacing along the helical axis from one base to the next. So per turn
there is 34 Angstroms. (3.4 A x 10 bases)
Sequence = information communicated in triplets (codons)
o Directionality 5’  3’
o The two strands are Antiparallel/Parallel ? (Circle)
o Consider some of the similarities between protein helix and dna, and some
differences.
o Rails made by sugar-phosphate backbone (antiparallel) and the Steps created by
purine-pyrimidine Hbonds
Nucleotide: sugar + base + 3 phosphates
Nucleoside: sugar + base
Sugar: deoxyribose vs ribose , what is the difference?difference is –OH group on the 2’
carbon
Bases: A, G, T, C -> rembember “AnGels are Pure)
o Which are purines and pyrimidines?
o How many hydrogen bonds?
Bond connecting the sugar to the phosphate is called what? Glycosidic linkage.
I.
Protein Folding: Process which the polypeptide chain acquires a correct 3-D structure, and a
biologically native state. (complex problem)
II.
Some proteins will fold into their native state spontenously, others require the assistance of
enzymes.
 Chaperones bind to partly folded proteins and prevent it from making illicit associations
with other folded or partly folded proteins – it also promotes folding of the protein it
holds.


After a protein has acquired most of its secondary elements, a loser tertiary state is
called the molten globular state -> this molten globule state will compact to form the
native state spontaneously.
How to predict the 3D structure of a protein is still an unsolved problem.
T/F -> a protein in its native state is static?
False, proteins in their native state are not static, there can be movements and changes and
“molecular Breathing”. Remember Induced fit for enzymes and things

Important: there tends to be only one biologically active fold in the native state this
native state has lower free energy than the others. -> there can however be other
folded states that are stable, but usually not biologically active.




III.
The two main difference between unfolded and folded states are enthalpy and
entropy.
Enthalpy derives from all the energy of the non-covalent interactions within the
polypeptide chain. (the covalent interactions do not change from unfolded and
folded proteins)
i. In the native folded state enthalpy is maximized and enthalpy is much larger.
ii. Therefore enthalpy is the driving force towards the _____folded_____ state
Entropy – measure of randomness, proteins in their native state are ordered and not
random, so their entropy is low. In the absence of other factors it would be more
favorable for the protein to be in the unfolded state, higher entropy (universe
prefers randomness).
i. Therefore the entropy is the driving force towards the unfolded state
Total difference between the enthalpy difference and entropy difference of the
protein is called the free energy difference. Usually it is very small/large ? (circle).
Because of the small free energy difference proteins are unstable and slight changes in pH
or temperature can convert proteins In their native state to the unfolded state.
Denaturants => cause large, structural change and loss of function
i. Usually cause abrupt loss of function -> protein unfolding is cooperative.
ii. Important- > do not break covalent
Denaturants will distrupt hydrophobic interactions. Eg. Urea.
1.)
2.)
3.)
4.)
DO NOT NEED TO KNOW ALL THESE
Heat -> alter hydrogen bonding.
pH extremes => changes ionization states -> causes electrostatic repulsion and effect HBonding.
Organic Solvents => disrupt hydrophobic interactions in the stable core. E.g. chloroform.
Detergents => disrupt hydrophobic interactions. E.g. SDS.
Christian Anfinsen Experiment : Proved that the 1° amino acid sequence contains all the
information required for proteins to fold into their 3-D conformation.




Studied what protein? Ribonuclease H,
Is this protein an enzyme? Why is it important to use an enzyme?
o He needed to be able to assay the proteins function, used an enzyme and
measured its activity.
Took a catalytically active protein added urea(what denaturant?) and
mercaptoethanol(reducing agent). -> caused what? -> loss of function.
o How did he regain the function? Used a dialysis membrane which filtered out
small molecules such as urea and B-ME. The protein then spontaneously
refolded into its original state which was again catalytically active.
Implcations
o
o
o
o
Protein sequence is determined by the aa sequence
Structure + function are associated
Under some conditions can be reversible
Under some conditions can be induced
Levinthal paradox. -> 150 residues, 3 configurations , each conf is tried in 10-12 picoseconds =
1048 years to fold.

What was the conclusion he made from this calculation? Folding is not random, it is
deterministic. Protein folding is deterministic. It must go through a kinetic pathway of
unstable intermediates to escape sampling a large number of irrelevant conformations.
Protein folding models
1. Molten Globule -> first, hydrophobic(fill) collapse -> spontaneously collapses into partly
organized globular state (less compact than native), driven by hydrophobic interactions.
Molten globule state may have 2ndary structure but it will not have the same
compactness.



I.
Q: is the formation of the molten globule fast or slow?
Q: is the molten globule just a single protein state?
Q: What is the second step in protein folding and what does it involve, is it slower or
faster?
The formation of the molten globule is very fast. Should not be viewed as a single
structure but rather an ensemble of many related inter-converting structures.
a. The second step is the slower step, can last up to 1 second, formation of
subdomains, Then it condenses and fixes the positions of secondary structures
and R groups
b. Secondary structure formation can not be the key event, it is the collapse of the
hydrophobic residues.
Free energy funnel
II.
a. Proteins can have more than one pathway to folding.
b. Represents the folding pathway of a protein as it assumes its native state.
c. The folding funnel hypothesis is closely related to the hydrophobic
collapse hypothesis, under which the driving force for protein folding is provided
by the stabilization associated with the sequestration of hydrophobic amino
acid side chains in the interior of the folded protein
d. The depressions on the side represent a semi-stable folding intermediate.
e. The width of the funnel represents the entropy of the protein.
Q: Have both single and multiple folding pathways been observed?
f. Both single and multiple folding pathways have been observed.
g. As the folding progresses the entropy of the protein decreases/increases
(circle?), but its enthalpy is increasing/decreasing (circle?). The difference is the
free energy, which decreases/increases? as the protein folds.
IV.
Protein Stability (thermal) What makes a protein more stable?


What makes a protein stable was discovered using a technique known as ?
o Site directed mutagenesis.
Reducing what tends to increase protein stability?
Reducing degrees of freedom (entropy) increases protein stability
1.) S-S bridges
a. -CH2-S-S-CH2b. Analysis of all possibilities (many)
c. Energy minimization to reduce to a few plausible candidates
d. Site-selective mutations
e. Protein synthesis
f. Assay:
example – T4 lysozyme (x-ray structure known)
2. Gly and Pro
-Gly  freedom
-Pro  Constraints (side chain is fixed by covalent bond to main chain
- Gly Pro has propensity to increase stability (more deficate)
- GlyAla usually increase
-Gly usually decrease
3. Dipolar stability
N-end has a _positive_____ dipole -> add what type of a.a. to increase stability? (-a.a.)
C-end has a _negative______dipole -> add what type of a.a. to increase stability? (+ a.a.)
4. Hydrophobicity in the core (cavity)
-Barnase (bacterial RNAse-110 a.a.)
-structure by both x-ray and NMR
-introducing cavities in the core by mutations such as IleVal or PheLeu ,Cavity for a CH2
-Stability by 1kcal/mol-More delicate design
-Needs structure
Obstacles to protein folding ( 3 main problems)
1.) Formation of incorrect __________ disulfide bonds.
a._________ protein disulfide isomerase -> catalyses the
shuffle/formation of covalent disulfide bonds until the native
conformation is formed. (Resident ER protein –> oxidizing enviorment.)
2.) Isomerization of _________ proline -> cis or trans.
a. ____________ Peptide Proplyl Isomerase-> Catalyses the
interconversion of cis/trans isomer of the proline peptide bond.
b. Proline has an unusually conformationally restrained peptide bond
due to its _______ (fill) cyclic stucture. Most amino acids have an
energetic preference for the trans peptide bond due to steric hindrance,
but proline's structure stabilizes the cis form so that both isomers are
possible.
3.) Aggregation of intermediates through exposed __________ hydrophobic
groups. i.e. hydrophobic clumping.
a. __________molecular chaperones -> inhibit inappropriate
interaction between complementary surfaces.
i. Bind to unfolded, partially folded, or incorrectly folded protein
-> facilitate correct folding pathways or provide appropriate
microenvironments for folding to occur.
b. Due to a very high concentration of proteins in solution.
2 classes of molecular chaperones.
1.)
Hsp70 -> (Heat___ Shock___ Protein____) -> abundant in cells stress
by ______high temperatures(fill).
a. Bind to unfolded poly peptides rich in _______ hydrophobic
residues(fill). Preventing inappropriate aggregation.
b. Bind to and release polypeptides in a cycle that uses
_________energy from ________ATP hydrolysis.
c. Also block the folding of proteins that must remain unfolded
until they become translocated across a membrane. Eg.
Proteins that are being translated across the ER membrane.
2.)
d. Highly conserved, different versions depending on cellular
location.
Chaperonins -> GroEL HSP 60/ES HSP 10
a. GroEL structure = Two heptameric (7 subunits each) rings ->
form two large independent pockets.
i. Hydrophobic patch on the opening (apical domain) binds
exposed hydrophobic regions on unfolded proteins.
b. GroES structure = Heptamer (7 subunits) -> Binds to and blocks
on of the GroEL openings in the presence of ATP.
c. Promiscuous -> bind and assist many proteins in folding,
independent of their a.a. sequence.
d. Gro EL: 3 domains -> equatorial, intermediate, and apical.
i. Equatorial: mainly helical and is an ATPase enzyme.
ii. Apical domain: alpha-beta sandwhich -> rich in
hydrophobic residues and are involved In binding to the
hydrophobic areas exposed by non-native folds of
polypeptide chains.
iii. Intermediate: small domain containing some alpha
helices that serves as hinges during conformational
change.
e. GroES cap: binding of GroES to one of the rings of GroEL will
decrease the affinity of the other GroEL ring for the GroES cap.
i. The core subunit is a beta barrel
ii. With two of the loops extending above the barrel
forming the roof hairpin which covers the center of the
dome.
iii. The other loop region Mobile Loop which is rich in
hydrophobic residues extends below the dome and
presumably interacts with the GroEL apical subunit.
GroEL/ES Mechanism. Look at the figure in the book as you read this it will help.
1.) Unfolded substrate proteins bind to a hydrophobic binding patch on the
apical domain of the open cavity of GroEL, in addition to 7 ATP causing a
conformational change.
2.) This conformational change allows for the binding of the GroES lid structure
-> causing the substrate protein to be ejected from the rim into the now
chamber.
3.) The microenvironment of the chamber favors the burying of hydrophobic
residues of the substrate, inducing substrate folding. After 13 seconds the
7 ATP are hydrolysed
4.) A second unfolded protein + 7 ATP bind to the opposite side causing the
other ring -> causes the release of the GroES cap plus better folded protein
+ 7 ADP
This cycle can repeat many times, and proteins and quickly go back into the
chamber.
DO NOT NEED TO KNOW ALL THE STUFF ABOUT PROTEIN FOLDING
DISEASES!!
 Proteins that remain unfolded are sent to the
______________proteasome(fill)
III. Diseases of protein folding
Amyloidosis -> a class of diseases involving extracellular deposits of insoluble
aggregates of normally soluble proteins.
 Soluble proteins that are normally excreted are excreted in the misfoled
state -> causes _______ (fill) amyloid plaques
 Native state of the protein does not exhibit __________ structural
similarity (fill)
 Continuous stacks of ________beta sheets, highly ordered (fill)
 How? Core beta-sheet forms before the rest of the protein folds correctly
and beta-sheets from two or more incompletely folded protein molecules
associate to begin forming an amyloid fibril.
 Other parts of the protein then fold differently, remaining on the outside of
the b-sheet core in the growing fibril.
 Often a high concentration of ________ aromatic, F, W, Y(fill) residues in
the core B-Sheet region.
 Beta strands -> stable alternative structure.
 Cystic fibrosis -> ___________________(fill) not caused by protein folding
Prion -> P______ I______ O_____ (fill) Proteinaceous Infectious Only
 Disease caused by a protein only-> no ___________ (fill) nucleic acid
 Degenerative brain disease -> e.g. __________________ bovine spongiform
encephalopathy, kuru, scrapie( sheep goat) creutefeldt Jacobs
(hereditary)(fill).
 Can spread to one organism to another. -> contagious (ingestion)
 Can be caused by a mutation
 Neurons develop large holes or vacuoles.
 Two forms -> 1.) ____________PrPc (cellular) 2.) _____________PrPsc
(scrapie)
o Normally Cellular form -> _____________ (fill) hydrophobic
membrane protein (signaling protein)o Gets converted into the abnormal form by contact (domino effect) ->
formations of __________ beta fibrils
o Important -> both forms have the same _____________a.a.
sequence
o Solution is a small molecule that could prevent fibril formation
Induced fit: proteins are not rigid, but rather undergo molecular breathing.
Paper 1
A. Voltage Gated Potassium Channel -> allows potassium to move down its electrochemical
gradient. Out of the cell under standard conditions.
a. S4 helices -> contain conserved arginine residues involved in voltage sensing.
i. S1-S4 is voltage sensing region -> cluster of positively charged A.A. Residues .