Protein Flashcards
(109 cards)
1
Q
What is a polypeptide?
A
A chain of amino acids
2
Q
How many amino acids are there?
A
20
3
Q
What is the structure of an amino acid?
A
Amino group (NH2). Carboxylic Acid (COOH). Hydrogen (H). R group.
4
Q
What do all alpha amino acids have?
A
Central carbon - alpha carbon
5
Q
What are all amino acids described as, apart from glycine?
A
Chiral
6
Q
What has to happen for different configurations of the same compound to occur?
A
Covalent bonds must be broken
7
Q
What stereoisomeric form do naturally occurring chiral centered compounds take?
A
D or L
8
Q
Which way does an L isomer rotate?
A
To the left. Follow CORN rotates clockwise.
9
Q
What are the different types of hydrophobic side chains?
A
Aliphatic (non-polar) and Aromatic.
10
Q
What are the different types of hydrophilic side chains?
A
Polar, uncharged (forms hydrogen bonds), positively charged (ionized) and negatively charged (acidic)
11
Q
What is the Equilibrium Constant (Kc) used for?
A
To determine the strength of weak acids
12
Q
What is the Kc equation?
A
[C]c [D]d
[A]a [B]b
13
Q
Why we use pKa values?
A
Ka values can be very small
14
Q
What is the equation for pKa?
A
pKa = -log10 Ka
15
Q
What would indicate a strong acid?
A
large Ka but a small pKa
16
Q
What is the Henderson-Hasselbalch equation?
A
pH = pKa + log10[A-]/[HA]
17
Q
Explain what the Henderson-Hasselbalch Equation means?
A
- WHne pH = pKa the concentrations of the protonated and depronated species are equal
- When pH < pKa, [HA]>[A-}
- When pH>pKa, [A-}>[HA]
- Basic amino acids have a high pKa and ten to bind protons
- Acidic amino acids have a low pKa and tend to release protons
18
Q
What does the isoelectric point indicate?
A
Is the pH where an ionisable molecule carries no net charge
19
Q
Why might a theoretical pI not match the experimental pI?
A
Sidechain of the folded protein is affected by its environment
20
Q
Why will different proteins have different optimal activities?
A
pH. The overall charge will change as protons are gained and released.
21
Q
What are the 4 levels of protein structure?
A
Primary. Secondary. Tertiary. Quartenary.
22
Q
Why bonds form between amino acids?
A
Peptide bonds
23
Q
What is the N terminus?
A
Start of the protein - refers to the free amine group
24
Q
What is the C terminus?
A
The carboxyl end of the protein
25
How are peptide bonds formed?
Proteins synthesised by ribosomes. Amino acids join during translation and form a peptide bond with the amino next to it.
26
What is the primary structure of a protein?
Sequence of amino acids
27
Why is the structure of the protein important?
Determines its function
28
Why does a polypeptide chain have polarity?
Because of the N terminus and the C terminus (+)
29
What type of bond does the peptide bond contain?
Partial double bond
30
What is usually the distance from C-N in a peptide?
1.32A
31
What is the expected distance for a single bond?
1.49A
32
What is the expected distance for a double bond?
1.27A
33
What are the bonds between N-Ca and Ca-C?
Pure single bonds (they can rotate)
34
Can double bonds rotate?
No, they are very rigid
35
What is a torsion peptide or dihedral angle?
The angle between planes through two sets of three atoms having two atoms in common
36
What are the 3 dihedral angles?
Omega, ⍵
Phi, Φ (N-Ca)
Psi, Ѱ (Ca-C)
37
What planar are the dihedral angles between?
-180 and 180 degrees
38
Why are the dihedral angles important?
Provide flexibility for the polypeptide backbone to adopt a certain fold. Due to the partial double bond of the peptide bond
39
What is the trans isomer in the protein?
Ca atoms on opposite sides of peptide bonds
40
What is the cis isomer in the protein?
Ca atoms on the same sides of peptide bond
41
Why is cis generally unfavorable?
You would get steric clashes eg benzene rings on amino acid would clash
42
Describe proline and what isomer it would be.
Secondary amino acid with a ring structure. Conformationally rigid. Would get steric clashes with the trans isomers so would be cis isomers
43
What does the Ramachandran Plot do?
Plots dihedral angles, Psi (y axis) and Phi (x axis). Rotates angles to see what is favourable
44
What does dark green represent in a Ramachandran Plot?
Allowed
45
What does light green represent in a Ramachandran Plot?
Partially allowed
46
What does no colour represent in a Ramachandran Plot?
Not allowed
47
What are the two exceptions from the normal in the Ramachandran plot?
1. Glycine - H side chain, more region would be green
| 2. Proline - favours the cis, restricted Ramachandran plot
48
Describe disulphide bonding in proteins.
Between Cysteines - lose 2 hydrogens, 2 sulphurs form a disulphide bond. Covalent. Singloe. Can connect 2 cysteines that are far apart in the primary structure.
49
What is the structure of keratin?
Helical. Two chains wrapped around each other.
50
Where is keratin found?
In nails and hair.
51
How does a polypeptide fold to form a more stable structure?
- Linear chain, not very stable
- Protein wants to fold to a lower energy structure
- Goes from linear to native
- Can fold via different routes and different intermediates
52
In what way is the native structure more stable?
Thermodynamically stable
53
What are the different forces that stabilize a proteins structure?
Covalent bonds. Electrostatic interactions. Hydrogen bonds. Hydrophobic effects.
54
Describe electrostatic interaction.
Two oppositely charged molecules. + and - attract. Distant depend, closer = stronger. The salt bridge (ion pair)
55
What are the different dipole-dipole interactions.
Permanent dipoles. Permanent dipole - induced dipole. Van der Waals.
56
Describe permanent dipoles.
Electronegativity. eg O is more electronegative than C and so attracts electron density.
57
Describe Van der Waals.
Strength dependent on distance. Interactions between instantaneous dipole and subsequently induced dipole. Too close = repulsion.
58
Describe Hydrogen Bondng.
Electronegative atom eg O or N, more electronegative than H so pull the electrons closer meaning H is slightly +vely charged. You get lots of these
59
Describe the hydrophobic effect
Fatty acids = hydrophobic chain disturbs the hydrogen bonding patter in water. Not as free to form H bonds. Become more ordered. Forms structure round non-polar - brings non-polar molecules together
60
Explain hydrogen bonding in alpha helix
Get hydrogen bonding between N-H and C=O in residues in one chain
61
Explain hydrogen bonding in beta sheets
Get hydrogen bonding between 2 polypeptide chains
62
Explain bonding in the alpha helix
Hydrogen bonding with chain
Optimal between C=O and N-H
Orientation of side chains away from centre of helix - right handed
Pitch = 5.4 Å - 3.6 residues per turn → distance between on section of helix to the next part aht repeats itself
Exceptions - glycine or proline
Proline wouldn't be able to form hydrogen bonding network - not in α helices
α helix has a net dipole movement
Lone par can move which induces dipole - N has a + charge
The helical wheel - side chains - one side chain contains charged and the other side contains hydrophobic parts - are some exceptions - important property
63
Explain bonding in beta sheets
Parallel and antiparallel → arrows show the direction between the N terminus and the C terminus
Parallel → same direction
Antiparallel → opposite directions
H bonds is between the sheets
In parallel H bonds at angle but not in anti parallel
Combination of parallel and antiparallel
Parallel structure can be formed - residues - can enclose things to transport etc
64
What are alpha helices and beta sheets joined by?
Turns and loops. In turns you can find proline residues
65
What is there between secondary and tertiary structure?
Intermediates
66
What is the tertiary structure?
Overall 3D shape of a protein
67
What is the Quaternary structure?
Spatial relationship of individual polypeptides within a protein complex
68
How can be proteins be described?
By the number of subunits they have known as subunit stoichiometry
69
What are the 3 methods of determining protein structure?
X-ray crystallography. Nuclear magnetic resonance (NMR) spectroscopy. Cryo-electron microscopy.
70
What is x-ray crystallography?
- Crystal of the protein
- xray beams diffracted
- Diffraction picked up by detector
- Different diffraction patters = different proteins
71
What is Nuclear Magnetic Resonance (NMR) spectroscopy?
- Sample dissolved in solvent
- Put in magnetic field and irradiated with short pulse of RF radiation
- Analyse the trace
72
What is Cryo-Electron Microscopy?
- Sample is frozen
- Electron microscope where it's bombarded with electrons
- Refined
- Can get a 3D map
73
What is an integral protein?
Span the whole membrane
74
What is an example of an integral protein?
Bacteriorhodospin, alpha helices - 7
75
What is a peripheral protein?
Not within in the membrane but still associated with the membrane
76
What is a Hydropathy plot?
Given the primary structure of an unknown protein - look at the properties of amino acids
77
What could polar/charged residues within transmembrane helices indicate/
Exposure to aqueous environment via channel formation. Membrane pore, transporter or carrier protein.
78
What is protein denaturation?
Loss of secondary and/or tertiary structure that causes loss of function
79
What are some examples of denaturants?
Strong acid or base. Alcohol. Thermal. All disrupt hydrogen bonds
80
Why is thermal stability important?
Proteins must remain folded at the temperature of that organisms environment to retain activity
81
What do chaperones do?
They are specialized protein complexes that help proteins to fold
82
What happens without chaperones?
Proteins misfold
83
What is the consequence of misfolded proteins?
They can form aggregates and plaques. This is a common characteristic of Alzheimers and Parkinsons
84
What did the Anfinsen Experiment show?
That the primary structure determines the tertiary structure.
85
What are cofactors?
Metals or coenzymes that assist in enzyme activity
86
What are coenzymes?
Small organic compounds
87
What is a prosthetic group?
Permanently associated cofactor
88
What are some examples of cofactors?
- Haemoglobin, heme group
- NAD, nicotinamide
- Zinc finger, zinc iron
89
What are some examples of covalent modification?
Glycosylation. Hydroxylation. Phosphorylation. Acetylation.
90
Describe Glycosylation
- Addition of a carbohydrate
- Can be O linked or N linked
- Asn-X-Ser/Thr (X is anything but Pro)
- Can add more than one sugar and get branching
91
What is meant by O linked in glycosylation?
Sugar attached to hydroxyl (OH) group = Ser, Thr
92
What is meant by N linked in glycosylation?
Sugar attached to the Amine group by nitrogen = Asn
93
Describe Hydroxylation
- Addition of OH group
| - Proline & Lysine
94
What is Hydroxylation like in Proline?
Very rigid. Side chain forms ring back with the amine group. If you hydroxylate becomes more rigid. Collagen is hydroxylated - contains proline.
95
Describe Phosphorylation.
- Addition of phosphate group
- Ser & Thr
- Have an OH group
- Changes the structure (locally or bigger effects)
- Protein could be phosphorylated and when you dephosphorylate thats its active form
- Reversible
96
What is Phosphorylation catalysed by?
Protein Kinase - Uses ATP, Phosphate transferred, Results in ADP
97
Describe Acetylation
- Transfer acetyl group to a lysine residue
- Catalysed by histone acetyltransferase
- Lysine = positively charged, acetyl negative, attach
- Reversible
98
What is the aim of Protein Purification?
Increase specific activity of your target
99
How would you identify how purified lactate dehydrogenase is?
- Use an assay
- Lactate and NAD+ catalysed by lactate dehydrogenase
- Forms pyruvate and NADH and H+
- Absorbance will be proportional to the concentration of NADH (absorbance of NAD+ and NADH are different)
- Can determine protein concentration
100
What is meant by activity?
Total units of enzyme activity
101
What is meant by specific activity?
Number of enzyme units per milligram of total protein
102
What is yield?
Total activity
103
What is purity?
Specific activity
104
On what properties are proteins purified?
Size. Charge. Hydrophobicity. Solubility.
105
What is Liquid Chromatography?
- Column contains matrix
- Sample at top
- Interacts with matrix X
- Move at different speeds due to level interaction
106
What is ion exchange chromatography?
Separate based on charge
107
What are the stages of ion exchange chromatography?
1. -ve matrix
2. Proteins which have a negative charge will flow thoguh
3. Positive ones will bind to the matrix
4. Can change salt concentration
5. Colour the proteins in order
108
What is size exclusion chromatography?
Separates based on size and shape
109
What are the stages of size exclusion chromatography?
1. Matrix has pores of certain sizes (cross-linked polymer)
2. Larger molecules pass straight through
3. Medium and smaller molecules get trapped in the pores
