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What are the 4 levels of protein structure?
- Primary: Amino acid residues
- Secondary: α-helix/β sheets
- Tertiary: Polypeptide chain
- Quaternary: Assembled subunits
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Protein molecule adopt what specific conformation, unlike most organic polymers?
Three-dimensional conformation
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The three-dimensional conformation is key to what in a protein?
The ability of protein to fulfill a specific biological function
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What is this three-dimensional conformation called where protein has activity?
Native fold
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What exists in large numbers within the native fold of a protein?
Favorable interactions
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What happens to entropy when folding the protein into one specific native fold?
- When unfolded, there's high entropy b/c there are many states it can possibly be in
- Once it settles into one state, it drops in entropy
- There is a cost in conformational entropy
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What bonds stabilize the folding of proteins?
Disulfide bonds between cysteine amino acids
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Which level of protein structure determines the 3D structure?
Primary sequence
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Which experiment proved that all that is needed to make a functional 3D protein is contained in the primary sequence?
Afinsen Experiment (1973)
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What was the Afinsen Experiment?
- Took a folded protein
- Denatured it with urea and β-mercaptoethanol → unfolded and inactive
- Rapidly diluted urea and β-mercap → got scrambled protein, incorrectly folded and inactive
- Slow dialysis/dilution → Renatured protein, properly folded and fully active
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What did teh Afinsen Experiment prove?
All that is needed to make a fully functional 3D protein is present in the primary sequence
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Which of the 4 levels of protein structures can be predicted based on the primary sequence?
- Secondary structure- α helices and β sheets
- But not tertiary structure
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How do we know cysteines aren't solely responsible for the specific pattern of folding?
- Cysteines are likely to make disulfide bonds
- But could make them wrong, with the wrong combinations
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What predicts the specific pattern of folding of proteins into the secondary structure?
Specific amino acid sequences
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What are the forces involved in the unfolded protein state?
- More H bonds with water, less with self (overall number of H bonds probably similar to folded)
- Salt bridges with itself in an environment of higher dielectric constant (weaker bonds, higher enthalpy)
- Exposes more hydrophobic residues to water (less entropically favored)
- Van der walls interactions present
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What are the forces involved in the folded protein state?
- More H bonds with itself, less with water (enthalpy doesn't change much, but increased entropy in water b/c free water molecules)
- Salt bridges with itself in an environment with a lower dielectric constant (stronger bonds, lower enthalpy)
- Less hydrophobic groups exposed (hydrophobic packing, more free water, entropically favored)
- Van der walls interactions present
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What are salt bridges?
Interactions between water and charged group
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How do salt bridges affect interactions between the groups of the protein?
- Water weakens interactions between charged groups
- Positive and negative charges interact with water instead of with each other
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What does high dielectric constant mean?
Substances that interfere with charges more
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What is the difference in bond strength between charged groups due to salt bridges in an environment with a lower dielectric constant vs. a higher dielectric constant?
- Higher dielectric constant- Salt bridges form stronger, more stable bonds btwn dielectric substance and charged group
- Lower dielectric constant- Salt bridges form weaker bonds btwn dielectric substance and charged group
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What kind of bond properties partially dictates the stucture of the protein?
Properties of peptide bonds
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What is tautomerization in a peptide bond?
- Peptide bond has ability to resonate
- Resonance of the lone pair from the amide nitrogen to the carbonyl oxygen allows for greater electron delocalization
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What about the peptide bond results a partion double bond nature?
Tautomerization
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What does tautomerization of peptide bonds result in?
- Planarity: Due to partial double bond nature of amide C-N linkage
- Formation of partial dipole moment: Due to charge separation
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How does the partial double bond nature of peptides affect structure?
- Doesn't allow roation around C-N bond
- Planarity
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In peptides, we find that R chains of two contiguous amino acids are usually in what kind of configuration?
Trans
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Why are R chains of two contiguous amino acids in a peptide usually in trans configuration?
To avoid steric clashing from cis configuration, das not favored
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What's the exception for steric clashing in peptide structure?
- Proline
- Clashes/uncomfortable either way
- No strong preference between cis and trans
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A polypeptide is made up of a series of _____ liked at α-carbons
planes
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How many bonds separate sequential α-carbons in a polypeptide chain?
3
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Which bonds can rotate in a polypeptide chain?
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What are the names of the angles that describe the rotation of dihedral angles in polypeptides?
- phi (Φ) = N-αC
- psi (ψ) = αC-N
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What are dihedral angles?
Angles between planes
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Why would any bond, other than the N-C peptide bond, be rotationally hindered?
- Depends on the size and charge of the R groups
- Some combinations of phi and psi are favorable, while others are not b/c of steric crowding of backbone atoms with other atoms in the backbone or side chains
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What are the angles of phi and psi for a fully extended polypeptide?
180°
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What does a ramachandran plot show?
- The distribution of phi and psi, dihedral angles that are found in a protein
- Common secondary structure elements
- Reveals regions with unusual backbone structure
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Primary structure dictates which other levels of protein structure?
Secondary and tertiary structure
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What are secondary structures?
Refers to a local spatial arrangement of the polypeptide backbone
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What kind of bonds do secondary structures mostly rely on?
Hydrogen bonds
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What are two common arrangements for secondary protein structures?
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How are α-helices formed/stabilized?
By H bonds between nearby residues
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How are β sheets formed/stabilized?
By H bonds between adjacent segments that may not be nearby
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Do secondary structures fulfill all hydrogen bonds internally or externally?
Internally
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What is an irregular arrangement of a polypeptide chain called?
Random coil
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How is the backbone of an α-helix held together?
By H bonds between the backbone amides of an n and n+4 amino acids
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A right-handed helix has about how many residues per turn?
3.6
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Describe the organization of α-helices
- Peptide bonds are aligned roughly parallel with the helical axiz
- Side chains point out and are roughly perpendicular with the helical axis
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How are peptide bonds aligned with respect to the helical axis?
Parallel with the axis
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How are side chains organized on an α-helix?
- Point out
- Roughly perpendicular with the axis
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Which amino acids generally don't exist in an α-helix?
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Why don't α-helices have the amino acid proline in their structure?
- Proline tends to break them (not covalently though)
- Can't make hydrogen bonds so the structure just gets weird
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Why don't α-helices have the amino acid glycine in their structure?
Glycine is too flexible to be stable in α-helices
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What are the two ways α-helices can twist?
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Where's the dipole moment in a peptide bond?
- Carbonyl O negative
- Amide H positive
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Which bonds in an α-helix have similar orientation?
Peptide bonds
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The α-helix has a large _________ dipole moment
macroscopic
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Negatively charged residues often occur near which end of the helix dipole?
Positive end
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What kind of residues often occur near the positive end of the helix dipole?
Negatively charged residues
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What are supersecondary structures/motifs?
Intermediate structures between secondary and tertiary structures
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How do supersecondary structures come about?
Result from packing specific adjacent secondary structure motifs
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What are some examples of supersecondary structure motifs?
- Coiled coils in keratin
- Superhelix in collagen
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What are coiled coils in keratin?
- α-helices wrapped around one another and then arranged parallel in length
- Forms a protofilament
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What are superhelices in collagen?
Chains twisted together forming a 3 stranded helix
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What is hair made of?
Coiled α-helices
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Why aren't coiled coils considered tertiary structures?
- They're well organized
- Only consist of α-helices
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How are hair dressers doing biochemistry?
- There are disulfide bonds in hair
- Disulfide bonds join α-helices
- Reduce with moist heat -> weakens H bonds
- Curl and then oxidize to form bonds again
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How do β sheet structures arise?
- Due to planarity of the peptide bond
- Tetrahedral geometry of the α-carbon
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How is the β sheet structure held together?
By hydrogen bonds between the backbone amides in different strands
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What is the orientation of side chains in β sheets?
Protrude from the sheet in alternating up and down directions
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What are the two orientations that β sheets come in?
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What are the H bonds like in parallel β sheets?
- H-bonded strands run in the same direction
- Resulting in bent H bonds (weaker)
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What are the H bonds like in antiparallel β sheets?
- H-bonded strands run in opposite directions
- Resulting in linear H bonds (stronger)
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How are β strands usually depicted?
As a "pointed blade"
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What are β turns?
Whenever strands in β sheets change direction
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How often do β turns occur?
Frequently
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How are 180° β turns accomplished?
- Over 4 amino acids
- Stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence
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How can secondary structures be assessed?
By circular dichroism
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What is circular dichroism?
- A molecule will absorb circularly polarized light differently depending on structure
- Left-handed vs. right-handed
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What kind of graph is used to measure circular dichroism?
Absorbance vs. wavelength (nm)
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What does the absorbance vs. wavelength plot for circular dichroism tell you?
- Simply how many of the type of structure there are
- Not exactly how they're structured
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What bonds are involved in tertiary and quaternary structures of proteins?
- Ionic bonds
- Hydrogen bonds
- Disulfide bonds
- Van der waal's forces
- Hydrophobic interactions
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What was the first protein whose tertiary structure was determined?
Myoglobin
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How was myoglobin's tertiary structure determined?
From X-ray studies by Kendrew and Perutz
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What is a chaperone?
A protein whose job it is to help other proteins fold correctly
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Why are chaperones necessary, even though Anfinsen Experiment showed that nothing else was needed for the protein to fold properly?
As proteins get larger, they have harder time folding
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What cool quaternary structure of a protein provides a specialized environment for other proteins to fold?
GroEL/GroES
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What is GroEL/GroES?
- Quaternary structure w/ a specialized environment for other proteins to fold in
- Double barrel structure
- 7 identical subunits per barrel
- 14 identical subunits total
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Where are all the subunits of GroEL/GroES made?
- In the cytosol
- And they somehow come together
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How do proteins fold so quickly and correctly?
Definitely not trial and error cuz that would take too long, trying to sample every option
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What is protein denaturation?
- Loss of 3D structure sufficient to cause a loss of function
- No secondary structure either
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How can proteins be denatured?
- Heat
- Extreme pH
- Organic solutes and solvents
- Detergents
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Protein denaturation is cooperative...
Yup
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What is proteostasis in cells?
- Continual maintenance of the necessary set of active cellular proteins
- Gotta keep the number of functional proteins the same
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Proteostasis requires regulation of what?
- Synthesis
- Folding
- Refolding
- Degradation
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What generally happens when systems regulating proteostasis malfunction?
Usually leads to disease states
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What are some pathways contributing to proteostasis?
- Polypeptide; folding intermediate; native protein; misfolded protein; aggregation; autophagy; peptide fragments
- Polypeptide; folding intermediate; aggregation; autophagy; peptide fragments
- Polypeptide; folding intermediate; remodeling; misfolded protein; aggregation; autophagy; peptide fragments
- etc. etc.
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What are two main families of chaperones?
- Hsp70
- Chaperonins (10-15% of proteins)
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Where is Hsp70 more abundant?
- In cells that are stressed by elevated temperatures
- Heat shock protein
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What does Hsp70 do?
Can help folding or prevent folding when needed
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What are some examples of chaperonins?
- E.coli- GroEL/GroES
- Eukaryotes- analog is called Hsp60
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What is amyloidoses?
Diseases in which there is deposit of insoluble amyloid fibers due to misfolding of proteins
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What are some examples of amyloidoses?
- Alzheimers
- Parkinson's
- Huntington's
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What are amyloid fibrils?
- High amt in cells
- Contribute to cell death
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What happens if proteostasis fails to destroy/autophage the aggregation of misfolded proteins?
Diseases
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What are prions and why are they dangerous?
- Normal proteins that exist in the brain tissue in all mammals in one very stable state that's not pathogenic
- Under certain circumstances, can form another stable structure of β sheets
- Because of β sheets, they aggregate
- This causes functional problems (in brain) so neurons die
- In the pathogenic form, prion proteins are hard to detect and extremely hard to denature or destroy
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What causes spongiform encephalopathies?
- Pathogenic prions
- PrPc → PrPsc
- They interact, causes PrPsc folding
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