Week 2 Flashcards
Ribonuclease is a small protein that contains ____ linked via _____
8 cysteins
Linked via 4 di sulfide bonds
T/F:
proteins fold to the lowest energy fold randomly
False
not random
Within the fold of a protein, where would you find hydrophillic and hydrophobic parts?
Hydrophobic want to bury in the centre
Hydrophillic will be exposed to water
Expell water
T/F:
Charged residues are only found inside the protein fold
False
Try to put them outside the protein fold
If they are inside they should try to be neutralized
T/F:
It is worse to bury a charged residue than expose a hydrophobic one
True
T/F:
Fibrous proteins are soluble
False
Insoluble
How do type I-III collagen assemble in comparison to type IV?
I-III= assemble in fibrils IV= laminar network, plates that stack on top of each other
Describe the structure of elastin
Cross-linked random coiled proteins Mesh that is oriented in a random way Provides elasticity to tissues Hydrophobic Insoluble Random coil Permits the protein to stretch and recoil Coils pack closely together, amino acids interact with each other
C terminals form a double di-sulfide bond
Lots of glycine, alanine, valine
What amino acids are commonly found in collagen?
Glycine
Alanine
Proline (special case where proline is in a helix)
Describe the structure of keratin
helical rods with globular N and C terminals (to allow them to interact with neighbouring chains)
Durable, unreactive
Hard alpha and soft alpha
Lots of cysteine and cross links through disulphide bonds
Coiled coil comes together to form protofilaments
Protofilaments form protofibrils
Lots of protofibrils packed into cells to form keratin
(curly hair= more cysteins)
What is a coiled-coil motif?
Bundle of right handed alpha helixes wound into a left handed super helix
Describe the structure of silk
Beta sheets
Hydrophobic (makes it feel smooth)
Alternating sequence of Gly and Ala
Sheets stacked on top of each other
Which two types of components give spider silk its properties?
Fibrion= provides structure Seracin= glue/matrix
Who discovered the sequence of insulin?
Frederick Sanger
1953
What is the first step to find the sequence of insulin?
Separate the chains
achieved with: extreme pH, 8M urea, 6M guanidine HCl, high sal concentration- usually ammonium sulfate. Then purify the chains
Oxidation with performic acid= prevents them reuniting again
Reducing agents= makes sure they are purified, allows them to go back once you remove them out of solution
What is the second step to find the sequence of insulin?
Determine which amino acids are present
Acid hydrolysis and ion exchange chromatography
o Boiled water and 6M HCL, break all the peptide bonds
o Runs elution profile in chromatography manner
o In different pHs, there will be different peaks indicating various amino acids (provides an idea of the ratios of the amino acids)
o Quantity/composition can be conferred
What is the third step to find the sequence of insulin?
- Identify N and C terminal Residues
o N terminal= free amino group
o C terminal= free carboxyl group
o These terminals can therefore react
o Determine the sequence using DNFB
o Helps to identify the number of distinct polypetides
o Found that there were 2 polypeptide chains
What is a DNFB Sanger Reagent?
➢ DNFB reacts with the polypeptide to form a yellow compound
➢ Run it on TLC places with standards
➢ Measure the different ratio that they can migrate on the TLC plates with different compositions of solvent and their colour as well
➢ Can quantify each
Explain Edman’s Degradation
By dropping the pH you can release one amino acid at a time and analyse
Can be used to identify a protein of known sequence
What is a negative about Edman’s Degradation?
Only works for up to 39 amino acids (not enough)
How do you sequence polypeptide chains that are bigger than 39 amino acids?
Need to break it down into smaller constituents
Sequence N and C terminus first
Use proteases to break them into smaller constituents
determine the sequence
put them back together
What type of technique can be used to identify post translational modifications of a protein?
Mass spectrometry
How does mass spectrometry work?
Identify the mass
and therefore identify the amino acid composition
Differences in amino acid sequence can be used to identify _______
evolutionary divergences
What is a motif?
Specific arrangement of several secondary structure elements
e.g. beta-alpha-beta motif
beta-hairpin motif
T/F:
protein synthesis is energy demanding
True
Each step of protein synthesis requires ____
energy in the form of ATP
There are __ amino acids with __ possible codons
20 aa
61 codons
= redundancy
Which two amino acids have a single codon?
Methionine (Met) and Tryptophan (Trp)
List the 5 stages of protein synthesis
Activation of amino acids Initiation of translation Elongation Termination and ribosome recycling Folding and post-translational processing
Where does protein synthesis occur?
On free ribosomes in the cytosol
What tells the protein where to go once it has been made ???
a sequence of amino acids
