Protein Sequencing Flashcards
Describe the primary structure of proteins
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The sequence of amino acids
- All atoms bonded to the amide are in the same plane due to rigidity from resonance
- The most favourable orientation has the C=O groups of the amino acids trans to each other
Describe the secondary structure of proteins
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Local folding of the protein
- Stabilised by non-covalent forces (H bonds)
- Common motifs are the alpha-helix and beta-sheet
Describe the alpha helix secondary structure
- 3.6 residues per turn
- Each NH hydrogen bonds to a carbonyl 4 amino acids away
- Side chains are on the outside
Describe the beta sheet secondary structure
- Each NH hydrogen bonds to the carbonyl of neighbouring segments
- Anti-parallel: hydrogen bonding is straight across
- Parallel: hydrogen bonding is diagonal
Describe the tertiary structure of proteins
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The folding of secondary structural elements into a 3D shape
- Stabilised by non-covalent forces (hydrophobic, H bonds, and electrostatic interactions)
- Stabilised by covalent forces (disulfide bridges) in Cys thiol groups
Describe the quaternary structure of proteins
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A collection of tertiary structures
- Ex., hemoglobin, which has 4 subunits
What is AAA?
Describe the 3 steps involved
Amino acid analysis (AAA): technique to determine the relative amounts of amino acids present in a peptide
- Acid hydrolysis of a peptide into aa (some are destroyed)
- Separation of aas by distance using ion exchange column (HPLC)
- Quantification of the separated amino acids by converting them into chromophores and measuring abduance by UV-vis
- Beer’s law: the intensity of absorption is proprtional to amount
Describe how the ion-exchange column works
- It is a small tube filled with insolube polymer and has permanent charges
- Separation is determined by ionic interactions and therefore overall charge
- Ex., in a negatively charged column:
- Negative AAs come out first as they are repelled
- Polar AAs with no net charge (don’t stick to hydrophobic regions)
- Non-polar AAs (stick to hydrophobic regions)
- Positive AAs
- Ex., in a negatively charged column:
Describe the two forms of ninhydrin
How does ninhydrin work to make the amino acid a chromophore?
Ninhydrin reacts with the amino acids to make Ruhemann’s purple (post-column derivatization)
- Via nucleophilic addition in acidic conditions
How is the imine produced by ninhydrin made into Ruhmann’s purple
- It is decarboxylated
- Observe that the side chain is lost as an aldehyde, so that regardless of the amino acid, the same chromophore is produced
Describe the 5 enzymes and their cleavage sites that are commonly used to hydrolyse proteins (for sequencing)
How does Edman degredation work?
- Allows us to do N-terminal sequencing
- It identifies the amino acid at the N-terminal and cleaves it
- Done using an automated sequencer and can do 50 amino acids at once before accumulation of side products interferes
Describe the first step of Edman’s degredation; adding PITC to the peptide
Describe the second step of Edman’s degredation; treating with a strong acid
Describe the third step fo Edman’s degredation; nucleophilic acyl substitution
Explain how DNFB/Sanger’s reagent was used for N-terminal identification before Edman
- DNFB/Sanger’s reagent reacts via nucleophilic aromatic substitution
- EWGs make the sp2 carbon sufficiently positive for this
Using Sanger’s reagent, how can we determine whether a Lys is on the N-terminal or not?
The side chains of lysine (amines of NH2) may also react
However, we can tell if Lys was on the N-term or not, as it would have two DNP groups if it was, but only one if it was not!
How does carboxypeptidase A work?
- Allows us to do C-terminal sequencing
- Carboxyypeptidase removes the C-terminal amino acids
- Also analysed by HPLC
