Cell and Molecular - Lecture One
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- Created on: 26-06-17 14:44
Name three diseases caused by protein misfolding
Alzheimer's, Parkinson's, Cancer
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Describe an alpha helix conformation
Spiral conformation in which every backbone NH2 group donates a hydrogen bond to the C=O group of an amino acid 3 to 4 residues earlier along the protein
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How many nm and residues does a full turn of an alpha helix span?
0.5nm and 3.6 residues
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Which amino acids have high alpha helix forming propensities?
Methionine, alanine, leucine, glutamate and lysine
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Which amino acid have low alpha helix forming propensities?
Proline and glycine
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Describe a beta-sheet conformation
A secondary structure formed by hydrogen bonds between adjacent beta-strands
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True or false - aromatic/hydrophobic residues are found in the middle of beta-sheets?
True
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Name some amino acids that are likely to be found in the middle of beta-sheets
tyrosine, phenylalanine, tryptophan, valine, isoleucine
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What is the role of the enzyme zymase?
It catalyses the conversion of sugar into ethanol and CO2
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Describe the structure of the Src tyrosine kinase
It's multidomain - it has a large kinase domain, a small kinase domain, and 2 regulatory domains - SH2 and SH3
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True or False - the small kinase domain is made up of mostly alpha helices?
FALSE - the small kinase domain is made up of mostly Beta-strands, and the large kinase domain is made up of mostly alpha helices.
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What are the three interactions between proteins?
1. Ionic interactions - between +ve and -ve ions
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How do peptides fold into a protein?
They fold so the hydrophobic amino acids are hidden inside the structure to form a hydrophobic core region, where as the polar side chains are found on the outside as they make hydrogen bonds with surrounding water molecules
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What is the primary structure?
The linear sequence of amino acids
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What is the secondary structure?
How the amino acids fold to give a particular shape e.g. alpha helix, beta-sheet or random coil
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What is the tertiary structure?
They way secondary structures pack together within a protein
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What is the quaternary structure?
The relationship between individual proteins into a multimeric complex
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How is the primary structure mainly inferred?
From the DNA sequence
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Name another way primary structure can be inferred?
By sequencing the amino acids
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What is the role of Edman degradation?
Used to work out the secondary structure of a protein by amino acid sequencing
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What is the role of mass spectrometry?
Used to work out the primary structure of a protein by amino acid sequencing
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Explain how Edman degradation takes place
It's a chemical disruption - PITC binds and disrupts the terminal amino acid, you then run HPLC to work out the MW of the amino acid.
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What is the main way in which secondary structure is predicted?
Use the primary sequence to predict secondary sequence - this can be done by de novo or by alignment with proteins of known structures
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Describe the structure of a coiled-coil?
Two amphipathic alpha helices wrap around each other so the hydrophobic sides come together in the centre and polar faces are exposed outways
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Where would you typically find coiled coils?
In elongated aggregated proteins
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Describe the structure of insulin
3 alpha helical elements and random coils
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What is the role of Circular Dichroism?
To estimate secondary structure
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What range on the UV spectrum is CD done in?
The far UV spectrum at 190-250nm
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On the CD spectrum, how many peaks would be characteristic of an alpha helix?
2
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On the CD spectrum, how many peaks would be characteristic of a beta-sheet?
1
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What is the definition of CD?
A differential absorption of circularly polarised light
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True or false - you can look at how various conditions affect protein structure e.g. pH and temp using CD?
True
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How does CD tell us about the stability of a protein?
Protein unfolding or melting is followed by CD at different temperatures, it causes the protein to uncoil, the less stable a protein the faster it loses its CD characteristics upon heating
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Describe how x-ray crystallography tells us about the structure of a protein?
A high energy focused beam of x-rays is fired through a protein crystal - look at the diffraction pattern to determine structural elements
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True or False - NMR uses isotopes such as C13?
True - the isotopes have different numbers of protons and neutrons and hence the nucleus has a slight wobble in the spin
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What is chemical shift?
Chemical shift is when different protons resonate at different frequencies depending on the local environment
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How would you introduce the isotopes into the structure of a protein?
You would produce them recombinantly in bacteria where the whole nutrient source is the isotopes, so all proteins are labelled
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True or false - NMR is a repetitive process?
T - you usually arrive at several possible structures represented as an ensemble
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What is TEM?
Transmission Electron Microscopy - protein is placed in heavy metal that electrons cannot penetrate, this is used to build up an overall structure
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Give an example of a heavy metal?
Uranyl acetate
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What is cryo-EM?
Proteins are frozen in vitreous ice, by shinning electrons onto the frozen structure you can determine the shape of the protein
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True or false - it is easier to process more radial symmetrical structures?
True - an example is a virus.
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Give three advantages of CD?
Its cheap, quick and theres no size limit
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Give two disadvantages of CD?
Limited dynamics and very low resolution (secondary structure only)
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Give three disadvantages of crystallography?
It's very costly, its slow and dynamics can be limited due to crystallisation artefacts
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Give two advantages of crystallography?
No size limit (although it gets harder as you get bigger) and it has the highest resolution overall - 1A
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Give four disadvantages of NMR?
Very costly, analysis is slow, isotopic label size can be a problem, size limit of 50kDa
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Give two advantages of NMR
Good dynamics, reasonable resolution - 2A
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True or False - EM is quite cheap?
False - it's quite costly
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Does EM have a size limit?
No
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Other cards in this set
Card 2
Front
Describe an alpha helix conformation
Back
Spiral conformation in which every backbone NH2 group donates a hydrogen bond to the C=O group of an amino acid 3 to 4 residues earlier along the protein
Card 3
Front
How many nm and residues does a full turn of an alpha helix span?
Back
Card 4
Front
Which amino acids have high alpha helix forming propensities?
Back
Card 5
Front
Which amino acid have low alpha helix forming propensities?
Back
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