1
Q
What makes up proteins?
A
Amino Acid Residues
2
Q
Why can it be difficult to annotate a gene?
A
o One gene several proteins
o Two genes overlapping
o One protein multiple functions
3
Q
How can proteins be post-translationallymodified?
A
- Phosphorylation (signalling)
- Glycosylation (protection, signalling)
- Proteolytic Cleavage (trafficking sequences)
- Acylation (fatty acids, localisation, regulation)
4
Q
Are proteins static? What is the best way to describe proteins in terms of motion?
A
No.
• Soft matter with flexibility
5
Q
What are the key things to remember about the property of proteins?
A
- One gene does not always equal one protein
- All cells have same DNA but not always same protein content
- Proteins are soft matter and both flexible with the potential to be highly structured
- Can be post-translationallly modified and their function can be changed
6
Q
What are the levels of protein structure?
A
Primary • Amino acid sequence from N-terminus to C-terminus Secondary • Local areas of regular ordered structure Tertiary • 3D fold of subunit Quaternary • Organisation of subunits
7
Q
Which way should an AA sequence be read in terms of terminals?
A
From N to C
8
Q
What is the general structure of an amino acid?
A
• L configuration, not D • α Carbon in middle with α hydrogen coming towards, carboxylate group (COO-), R group (R), Amine group (NH3+)- CORN • Zwitterionic: deprotonate • Amino Acid residue: -HN-CHR-CO-
9
Q
What is zwitterionic?
A
o neutral with no net charge at neutral pH. Both carboxylate and amine can be ionised
o Add protons (more acid), lower pka/pH, protonate
o Remove protons (more base), rise pKa/pH,
10
Q
What is L configuration?
A
can be synthesised from L-glyceraldehyde
11
Q
How can groups be protonated or de-protonated?
A
Add acid (protonate, make more negative, lower pH and pKa, NH3+ and COOH)
Add base (deprotonate, make more postive, raise pH and pKa, NH2 and COO-)
12
Q
Can NH2-(CHR)-COOH exist? Why?
A
No. It’s not zwitterionic. No charges to balance.
13
Q
How do pH and pKa interact?
A
- pH = pKa + log [A-] / [HA]
- When [A-] = [HA], pH = pKa (log1 = 0)
- pH = pKa : [COOH] = [COO-]
- pH > pKa : [COOH] < [COO-] more acid
- pH < pKa : [COOH] > [COO-]
14
Q
Where on a protein will hydrophobic or hydrophilic residues be found?
A
- Hydrophobic : inside protein
* Hydrophilic: outside protein
15
Q
Which amino acids are hydrophilic (charged, polar)?
A
• Acidic (-charge at neutral pH, low pKa, carboxylate) o Aspartate (Asp, D) o Glutamate (Glu, E) • Basic (+charge at neutral pH, higher pKa) o Lysine (Lys, K) o Arginine (Arg, R) o Histidine (His, H)
Hydrophillic = charged and polar
16
Q
Which amino acids are hydrophilic (neutral, polar)?
A
• Carboxamide o Asparagine (Asn, N) o Glutamine (Gln, Q) • Hydroxyl o Serine (Ser, S) o Threonine (Thr, T)
17
Q
Which amino acids are hydrophobic (aliphatic)?
A
- All methyl groups
- Alanine (Ala, A)
- Valine (Val, V)
- Leucine (Leu, L)
- Isoleucine (Ile, I)
- Methionine (Met, M)
18
Q
Which amino acids are hydrophobic (aromatic) ?
A
• Phenyl o Phenylalanine (Phe, F) • Phenol o Tyrosine (Tyr, Y) • Indole o Tryptophan (Trp, W)
19
Q
Which amino acids fit the ‘other’ category?
A
• No R group o Glycine (Gly, G) • Thiol o Cysteine (Cys, C) • Pyrrolidine o Proline (Pro, P)
20
Q
Which AA residues are sometimes phosphorylated?
A
Serine, Threonine, Tyrosine
21
Q
Which AA residues are sometimes glycosylated?
A
Asparagine, Serine, Threonine
22
Q
What is involved in phosphorylation?
A
- Enzymatic addition of group
- Add phosphate
- Regulation/amplification of biological processes
- Changes chemical nature (polar neutral to polar negatively charged)
23
Q
What is involved in glycosylation?
A
- Enzymatic addition of group
* Add carbohydrate
24
Q
What are the features of aromatic groups?
A
flat, share double bonds, sp2
25
What group is common to hydrophobic (aliphatic) amino acids?
Methyl group
26
What are the branched chain amino acids?
Valine, Leucine, Isoleucine
27
What is the largest and rarest amino acid?
Tryptophan
28
Which AA lacks an R group?
Glycine
29
Which AAs absorb UV light well?
Tryptophan, Tyrosine
30
Which AAs are most common?
Alanine, glycine, leucine
31
What is the mean MW for AA residue?
110
32
What are the features of the peptide bond?
Stable but can be hydrolysed by proteases
| Partial Double Bond Character
33
What reaction forms a peptide bond? What molecule is lost during it?
* Condensation reaction between COO- and NH3+
| * Lose water
34
What forms the covalent structure of a protein?
Primary AA sequence
35
What are the features of the double bond character?
* Resonance structures (C=O, C=N)
* 1.32 A length
* Gives rigidity and planarity
36
Which configuration (cis/trans) is favoured with peptide bonds and why?
Trans orientation (180)
• Four atoms of relevance: Cα(i), Cα(i-1), N(i), C (i-1)
• More stable peptides are trans (avoid steric clash)
• Α C’s on opposite sides of bonds
37
What is the general rule to calculate molecular weight of a protein?
number of residues X 110 = Molecular Weight
38
What is isoelectric point?
• pH where protein carries NO charge
39
What is the net charge of a protein when pH < pI?
Postive
40
What is the net charge of a protein when pH > pI?
Negative
41
How does the length of a peptide influence pKa and ionisation?
• Longer peptide = more COOH and less NH3+
o Favourable reaction between NH3+ and COO- reduced due to distance
o COO- more remote and not free to stabilise NH3+
o Therefore the distance between pka1 (COOH) and pk2 (NH3+) decreases
42
What determines if a pI is acidic or basic?
* Large pI = basic
| * Small pI = acidic
43
How can we work out the sequence/covalent structure of a protein?
* Edman degradation (remove AA and identify)
* Mass spectrometry
* DNA sequence but won’t show post translational modification information
44
What is a torsion angle?
Dihedral angle
• Made up of four atoms, three bonds
• Counter clockwise = +ve
• Each peptide bond plane can be orientated to succeeding/preceding peptide bond planes by the two torsion angles φ and ψ
45
What is ω?
* Torsion angle of peptide bond
* Relevant atoms: αC (i-1), C’(i-1), N(i), αC(i)
* Cis: ω= 0
* Trans: ω= 180
46
What is φ?
* Rotation around N- αC bond
* No rotation around rigid peptide bond
* Relevant atoms: C’(i-1), N(i), αC (i), C’ (i)
47
What is ψ?
* Rotation around αC-C’ bond
* No rotation around rigid peptide bond
* Relevant atoms: N(i), αC (i), C’ (i), N (i+1)
48
Which angles characterise each residue in a protein?
Each residue ion protein can be characterised by a φ and ψ pair
49
How can φ and ψ interact? What do steric clashes prevent?
• Not completely free to rotate (steric clashes restricting)
50
What would φ and ψ angles of 180° result in?
No steric clash
51
What would φ and ψ angles of 0° result in?
Steric Clash
52
What kind of angle is favoured for φ? Why?
Favour φ to be negative
| • Side chain opposite (no clash between side chain and carbonyl oxygen)
53
What does the Ramachandran Plot show? What are the most favoured regions?
* Certain angles of φ and ψ favoured
* Favoured regions: α and β
* α (α helices)
* β (β sheets)
* Forbidden regions: steric hindrance
* Expect majority of structures in favoured regions
54
How can glycine be on the right of the Ramachandran Plot?
No R group, no steric hindrance if + φ
55
How does regular and irregular secondary structure differ?
Regular Secondary Structure
• Repeating φ and ψ angles for sequential residues
• Elements with repeating φ and ψ in α region =α helices
• Elements with repeating φ and ψ in β region =β strands (make up β sheets)
Irregular Secondary Structure
• Non-repeating φ and ψ angles for sequential residues
• E.g. β turn (different angles between sequential residues)
56
What does 3.613 mean?
• 3.6 residues per turn (one every 100°), 13 atoms in H bond ring = 3.613
57
What are the features of α helices?
* Side chains pointed away from helix
* Right handed
* φ -60° and ψ-50°
* NH and C=O in helix form internal favourable hydrogen bonds
* Carbonyls oppositely aligned to NH
* Amphipathic
58
In αhelices, what kind of bonds are formed?
• NH and C=O in helix form internal favourable hydrogen bonds
59
What does it mean for an α helix to be amphipathic?
o One side hydrophobic, one side hydrophilic
| o Helical wheel
60
What are the features of a β sheet?
* Made from β strands
* Strands = parallel or antiparralel to neighbours
* φ -130° and ψ +130°
* H bonds between C=O and NH of opposing strands
* Outer NH and C=O not H bonded
61
What are β turns? What kind of structure are they?
* Irregular stricture
* Reverse main chain
* Mostly on surface
* Four residues (different φ and ψ angles for i+1, i+2)
* Stabilised by H bond between Carbonyl of i and NH of i+3
* i+1 often proline
* i+2 often glycine in type II
62
How are type I and type II β turns different?
Type II
• i+2 R group on same side as i+1 (+φ)
• Expect clash
• Therefore i+2 Glycine because no R group, just H
63
What was the Anfinsen experiment?
* Showed AA sequence determines 3D structure
* Unfold protein in urea and reducer (ribonuclease A)
* Remove urea and reluctant, notice spontaneous refolding and correct disulphides reforming (expect 105 due to 8 cysteine’s, but only see 1)
* Remove reluctant and keep urea, random disulphides form
* Protein folds so in can position cys residues correctly
* Weak non covalent interactions that manoeuvre protein so disulphides can form
64
What is the basis of Van der Waals interactions and what is Rm?
* Inside of protein = compact
* Two atoms approach each other until reach VDW radii distance (favourable attraction up until the distance)
* Contact distance = energy minimum = Rm
* VDW radius varies for atoms (e.g. carbon is 1.7 Å, VDW distance between two carbon is about 4Å)
65
What are hydrogen bonds?
* Two electronegative atoms compete for same hydrogen
* Donor (D) δ+
* Acceptor (A) δ-
* Electrostatic interaction
* Transfer of electrons from A to H has covalent component
66
What helps proteins be compact?
Hydrogen bonds (shorter than VDW)
67
What length of hydrogen bond would be strong and mostly covalent?
2.2-2.5 Å
68
What length of hydrogen bond would be moderate and mostly electrostatic?
2.5-3.2 Å
69
What length of hydrogen bond would be weak and electrostatic?
3.2-4.0 Å
70
Where can hydrogen bonds occur?
* Backbone-backbone
* Backbone-side chain
* Side chain-side chain
* Polar groups buried in protein must form hydrogen bonds
71
What are salt bridges? What type of interaction are they?
* Electrostatic interactions
* Ionic interactions between oppositely charged groups
* Stabilises ionic interaction
* Charges distributed
* pKa for residues different to typical value (bases more, acids less)
72
What are hydrophobic interactions?
• In aqueous environment, hydrogen bond and VDW interactions between polar groups no favoured (competition with water)
• Proteins fold
o Water poor solvent for nonpolar groups (unlike organic solvents)
o Nonpolar groups cant form hydrogen bond networks
o Nonpolar groups prefer interact with other nonpolar groups
73
What drives hydrophobic interactions?
Entropy, solvent entropy = driver
| • Favourable/negative ∆G given if negative ∆H and positive ∆S (↑disorder)
74
How does entropy differ between unfolded and folded proteins?
```
Low Entropy +∆G
• Unfolded
• Lots of ORDERED water molecules
• Exposed nonpolar side chains
High Entropy -∆G
• Folded
• Bury non polar side chains
• Release ordered water molecules
• Disorder increases
```
75
How does ∆G relate to protein folding?
∆G = ∆H - T∆S
• Unfolded ↔ Folded (preferred)
• ∆G = ∆H - T∆S
• ∆H: VDW and H bonds (-15 - -20kj/mol)
• ∆S: disorder of protein and solvent entropy
• ∆G: G(folded) – G(unfolded)
• Native state proteins = marginally stable
76
Which methods are used to determine protein structure?
* X-ray crystallography (3D crystals)
* NMR spectroscopy (solution, solids)
* Electron Microscopy (2D crystals)
77
What are the ways to view and represent protein structure?
```
• Wire – line, stick
o Full details on position of atoms
• Cartoon – Ribbon
o Helix (coil) and strand (arrow) orientation and position
• Sphere
o Surface nature
o Residue exposure
```
78
What are the features of Myoglobin as a globular protein?
* Small, oxygen binding
* Eight α helices (different lengths, loops connect them or some β turns)
* Well defined ordered structure
* Most hydrophobic residues inside
79
What kind of bonding can be found in Myoglobin and where?
* Close contacts (VDW) between hydrophobic residues of helices
* No main chain hydrogen bonds between helices (H bonds within helices or with side chains)
80
What is the significance of the edge of Heme being exposed in myoglobin?
• Edge of heme exposed in order for oxidation to occur
81
What are the super-secondary structures?
* Repeating elements
* α-α corner/hairpin
* β-β hairpin
* βαβ element
82
What are the four rules of protein structure?
1. Hydrophobic interactions for stability. Need at least 2 layers of secondary structure fo H20 to be excluded and hydrophobic side chains to be buried.
2. α helices and β sheets in separate layers because backbone H bonding disallows helices to H bond to sheets.
3. Protein segments adjacent in sequence usually adjacent in structure.
4. β strands favour right hand twist and between two parallel strands. Connections shorter than left hand.
83
What kind of connections between β strands are shorter (left or right hand)?
Right Hand
84
What is the significance of proteins in the sequence usually being adjacent in structure?
* Some exceptions
* Non-random distribution of elements
* Distant regions still brought together
85
Why is the right handed β strand connection preferred?
* Need less protein to form connection
| * Conserve protein is favourable
86
What is a domain? What type of core does it have?
* Domain is region in tertiary structure of which evidence provides an existence independent of the rest of the protein
* Hydrophobic core in each domain separates them from each other
* Arrangement and order of secondary structural elements that contain single hydrophobic core
87
What are the features of domains?
* Size varies
* Individual domains: individual functions possible (catalytic, binding)
* Modular proteins, repeating domains, slightly different sequence, same fold
* Most proteins multiple domains (teamwork)
* Same protein domain can be found in functionally distinct proteins
* Same shape, sequence can vary
* So far identified 1500 unique domains
88
Why is it more accurate to describe a domain as an evolutionary unit rather than structural unit?
* The way new proteins arise suggests common protein ancestor
* Individual domains further subjected to mutation, insertions, deletions, randomly
* NO new domains created
89
How can protein domains be aligned?
* Based on residue identity or similarity
* Identity: exactly same residue
* Similarity: change to residue observed frequently or with similar phys-chem properties (ser to thr)
* Improve alignments with gaps
90
What are the reasons for gaps in protein domain alignment?
o Account for insertion, deletion of residues
91
What are homologues?
o Share common protein domain ancestor, share same fold
| >25% similarity = homologous
92
What are orthologues and paralogues?
Orthologues
o Homologous proteins, same function, different species
Paralogues
o Homologus proteins, different but related functions, one organism
93
How does protein structure compare for proteins that are related but have different sequences?
* Sequence change = small structure change
* Structure change much SLOWER than sequence change (sequences with >25% identity very similar structure)
* Similar secondary and tertiary structure
* Gaps = loop insertion within general structural fold
* Different parts of protein mutate at different rates (conservation of functional residues)
94
How can new proteins with new functions be generated?
* Mix domains and mutate existing domains
* Intragenic mutation (point, indel)
* Gene duplication
* DNA segment shuffle (broken, recombined)
* Gene lateral transfer
BCMB20002 Biochemistry
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