week 5 no answers

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Freyr Petursson TA handout, week 5, BIBC 100, [email protected], Office hours:
Tuesdays after class at the S and E library main floor. You can come to office hours
for answers.
I.
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II.
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Double stranded Base-paired Helical DNA introduction
____________________ -> obtained in vivo when DNA is felly hydrated.
_____________________ -> Dehydrated conditions (lab)
______________________ -> Left handed DNA
B-DNA structural Features.
B-DNA Diameter: _____________________. (how many angstroms)
o Right-handed helical “staircase”
o ____ bases per Turn , so a base every ______(degrees?)
o ________ is the spacing along the helical axis from one base to the next. So per turn
there is _________________ (How many angstroms)
Sequence = information communicated in _____________ (codons)
o Directionality : ________________
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 ___________________ (antiparallel) and the Steps created by
__________________________
______________________: sugar + base + 3 phosphates
________________________ sugar + base
Sugar: deoxyribose vs ribose , what is the difference?
Bases:
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 _________________, others require the assistance of
_______________________.
 ______________ 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
___________________ state -> this molten globule state will compact to form the native state
spontaneously.
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How to predict the 3D structure of a protein is still an unsolved problem.
T/F -> a protein in its native state is static?
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Important: there tends to be only one ___________________ 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.
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III.
The two main difference between unfolded and folded states are ________ and __________
__________ 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 __________ state
_____________– 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 __________________ state
Total difference between the enthalpy difference and entropy difference of the protein is
called the _____________________________. 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. _____________________?
___________________________? Experiment : Proved that the 1° amino acid sequence contains all the
information required for proteins to fold into their 3-D conformation.
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Studied what protein?
Is this protein an enzyme? Why is it important to use an enzyme?
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Took a catalytically active protein added ________(what denaturant?) and
_____________(reducing agent). -> caused what? ->
How did he regain the function?
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Implcations
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Protein sequence is determined by the _______________
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Structure + function are associated
Levinthal paradox. -> 150 residues, 3 configurations , each conf is tried in 10-12 picoseconds = 1048 years
to fold.
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What was the conclusion he made from this calculation?
Protein folding models
1. Molten Globule -> first______________________(fill) collapse -> spontaneously collapses into
partly organized globular state (less compact than native), driven by ____________________
interactions. Molten globule state may have 2ndary structure but it will not have the same
compactness.
 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?
I.
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 width of the funnel represents the entropy of the protein.
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Q: Have both single and multiple folding pathways been observed?
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 ?
 Reducing what tends to increase protein stability?
1.) ___________________________ ( hint: it involves sulfur)
b. 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. ________________________________( these a.a. have special properties)
-Gly 
-Pro 
- Gly
- Gly
-Gly
3. Dipolar stability
N-end has a ______ dipole -> add what type of a.a. to increase stability?
C-end has a _______dipole -> add what type of a.a. to increase stability?
4. ___________________________________(core of the proteins is what?)
OBSTACLES TO PROTEIN FOLDING.
1.) formation of correct ________________ bonds.
a._________ -> catalyses the shuffle/formation of covalent disulfide bonds until the native
conformation is formed. (Resident ER protein –> oxidizing enviorment.)
2.) Isomerization of _________ -> cis or trans.
a. ____________ -> 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)
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 __________ _______groups. i.e. hydrophobic
clumping.
a. ______________________________ -> inhibit inappropriate interaction between
complementary surfaces.
1. 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.)
(fill).
2.)
Hsp70 -> (H_______ S________ P________) -> abundant in cells stress by ___________
a. Bind to unfolded poly peptides rich in _______ _______(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.
d. Highly conserved, different versions depending on cellular location.
Chaperonins -> GroEL HSP 60/ES HSP 10
a. GroEL structure = Two ___________________ rings -> form two large
independent pockets.
i. Hydrophobic patch on the opening (apical domain) binds exposed
hydrophobic regions on unfolded proteins.
b. GroES structure = __________________________ -> Binds to and blocks on of
the GroEL openings in the presence of ATP.
c. ___________________ -> bind and assist many proteins in folding, independent
of their _________________________( fill)
d. Gro EL: 3 domains -> __________________, __________________, and
__________________l.
i. Equatorial: mainly helical and is _________________
ii. Apical domain: alpha-beta sandwhich -> rich in ________________ and
are involved In binding to the ____________________exposed by nonnative folds of polypeptide chains.
iii. Intermediate: small domain containing some alpha helices that serves
as _______________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 ______________
ii. With two of the loops extending above the barrel forming the
_____________ which covers the center of the dome.
iii. The other loop region ________________ which is rich in hydrophobic
residues extends below the dome and presumably interacts with the
GroEL apical subunit.
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