Proteins Flashcards
(188 cards)
1
Q
What are enzymes function?
A
Catalyse covalent bond breakage or formation
2
Q
What are the function of structural proteins
A
Provide mechanical support to cells and tissues
3
Q
What is the function of motor proteins
A
Generate movement in cells and tissues
4
Q
What is the function of signal proteins?
A
Carry extra cellular signals from cell to cell
5
Q
What is the function of transport proteins?
A
Carry small molecules or ions
6
Q
What is the function of gene regulatory proteins?
A
Bind DNA to switch genes on or off
7
Q
What is the function of receptor proteins?
A
Detect signals and transmit them to the cells machinery
8
Q
What is the function of special purpose proteins?
A
Their function is highly variable. For example proteins can act as messengers, antibodies, pumps and channels
9
Q
How do proteins differ?
A
- Chemical composition (amino acid sequence)
- Structure / shape
- Functions e.g. transport, structural, enzymatic etc
- Name (haemoglobin, insulin)
10
Q
What are proteins made of?
A
- Alpha amino acids
- Amino acid are linked end to end by covalent bonds in linear chain
- Amino acid chain known as a polypeptide
- Polypeptide is folded into specific 3D shape
- One or more folded polypeptides = protein
11
Q
What is the primary structure of an amino acid?
A
Linear amino acid sequence
12
Q
What is the secondary structure of a protein?
A
Polypeptide folding into regular shapes
13
Q
What is the tertiary structure of a protein?
A
Tertiary structure - arrangement of one/ multiple domains
3D structure
14
Q
What is the quaternary structure of a protein?
A
Arrangement of multiple polypeptides
Protein complex (multi-subunits)
15
Q
General formula for amino acids?
A
R-CH(NH2)COOH
R = side chain
COOH = carboxyl group
NH2 = amino group
C = alpha carbon
H = alpha proton
16
Q
What differs between the 20 naturally occuring amino acids in nature?
A
Side chains (R group)
17
Q
What is an amino acids at PH 7?
A
Zwitteron - it has both positive and negative charges
18
Q
Which is the only amino acid that is not chiral? Why?
A
Glycine
The side chain of glycine is H
19
Q
Isomers of amino acids?
Which is found in proteins in nature?
A
L and D
L are found in proteins in nature
20
Q
Which are ‘small and simple’ amino acids?
A
Glycine and alanine
21
Q
Which amino acids have hydrophobic side chains?
A
Valine (Val, V)
Leucine (Leu, L)
Isoleucine (IIe, I)
Methionine (Met, M)
22
Q
What can the amino acids Cystetine do?
A
Thiol group (-SH) can form disulphide bonds (S-S)
23
Q
Which amino acids have aromatic (ring) side chains?
A
Phenylalanine (Phe, F)
Tyrosine (Tyr, Y)
Tryptophan (Trp, W)
24
Q
Which amino acids have polar side chains?
A
Serine (Ser, S)
Threonine (Thr, T)
(Contains -OH group)
25
Which amino acids are positively charged / basic?
What property does this give them?
Lysine (Lys, K)
Arginine (Arg, R)
Histidine (His, H)
Hydrophilic
26
Which amino acids are negatively charged / acidic?
Aspartate (Asp, D)
Glutamate (Glutamate, E)
27
Which amino acids are charged?
Aspartate (Asp, D)
Glutamate (Glu, E)
28
What is different about the amino acid proline?
- Side chain forms a five membered ring by bonding to the nitrogen atom
- It is an Imino acid NOT amino acid
- Proline side chain is often rigid and forces a sharp ‘bend’ in the main chain
29
What bond are amino acids joined together with?
Where is the bond?
Covalent
Carbonyl group of one amino acids joins together the amino group on the next
30
What type of reaction is polymerisation?
What is the reverse?
Condensation reaction
Reverse = hydrolysis
31
What bond is between the C and N of two different amino acids in a polypeptide?
Peptide
32
When / where do disulphide bonds occur?
- Between side chains of two cysteine under oxidising condition
- Tend to be found in proteins that are present extracellularly
- Disulphide bonds can b cleaved by reduction with a reducing agent such as B-mercaptoethanol
33
What can effect an amino acids properties?
Size
Charge
Hydrophobicity
Polar / non-polar
34
What is sickle-cell anaemia caused by?
-Mutation that affects single amino acids joins together residue in haemoglobin B
- Glu and Val mutation
35
What is cystic fibrosis caused by?
Deletion of single amino acid (Phenylalanine) in te transmembrane conductance regulator (CFTCR)
36
What is the configuration of the majority of peptide bonds?
Trans
37
What does trans configurations allow for peptide bonds?
Allows amino acid side chain to be spaced far apart meaning no/less steric hinderance
38
What can happen when peptide bonds are in cis configuration
Steric hinderance
39
What is steric hinderance?
The repulsion or resistance that occurs when atoms or groups within a molecule are positioned close together. This can prevent chemical reactions from taking place / affect the reactivity
40
What is different about X-proline?
-X can be any amino acid
-steric clashes occur in bot cis and trans configurations
-X-proline bond can be present in cis more frequently than any other peptide bond
41
Why are atoms in peptide units in the same lane? How many atoms?
Six atoms are in the same plane due to the double-bond characteristics of the peptide bond
42
Which peptide bonds are rigid in the peptide units?
C-N peptide bond
43
Which bonds in peptide units can rotate? What does this mean?
C alpha - C
N - C alpha
44
What is rigid in a polypeptide backbone?
Peptide bond and amide plane
45
Which bonds are free to rotate in a polypeptide backbone?
N - C alpha
C alpha - CO
46
What is Phi ø?
Torsion angle of N - C alpha bond
47
What is psi?
Torsion angle of C alpha - CO
48
What does the conformation of the polypeptide backbone depend on?
Phi and psi
49
What are phi and psi restricted by?
Steric constraints:
-size of side chain (R)
-planarity of peptide unit
50
Why is the peptide backbone only partially flexible?
Only a certain combination of phi and psi are energetically favoured (stable)
51
Which angles are favoured in secondary structures?
B sheets and right handed alpha helix. These are on the right hand side of the ramachandran plot
52
What can protein structures be stabilised by?
Non-covalent interactions
53
Are non-covalent bonds stronger or weaker than covalent bonds?
20-100 times weaker
54
Three non-covalent bonds?
1- hydrogen bonds
2- salt bridges (electrostatic interaction)
3- van der waals’ interactions
55
What is a hydrogen bonds?
- Non-covalent bond that occurs when a donor atom donates its covalently bonded H atom to an electronegative acceptor atom
- H-bond is strongest (most stable) when the acceptor, hydrogen and donor lie in a straight line
56
When does hydrogen bonds occur in proteins?
H = partial positive
O, N = partial negative
57
What are hydrogen bonds needed for in proteins?
Crucial for folding and formation of secondary structure
58
What does salt bridges form from?
Oppositely charged atoms
59
Which amino acids form salt bridges?
Positive:
- arginine
- lysine
Negative:
- glutamic acid
- aspartic acid
60
What is van der waals’ interactions?
A non specific attractive force when two atoms are 3-4 Å apart
61
What do van der waals result from?
Transient shift in electron density
62
What happens when two atoms get too close (van der waals)
Repulsion
63
What happens to NH and CO is the backbone?
Neutralise each other via H bonds
64
What does the polypeptide form in an alpha helix?
Regular right handed spiral structure
65
How many amino acid residues form from five turns of the helix?
18 amino acid residues
66
What is the vertical rise per residue in an alpha helix?
0.15nm
67
How to calculate vertical rise per turn?
Number of residues per turn x vertical rise per residue
68
Length of helical stretches of polypeptide?
4-40 amino acid residues in length
69
What is the length of a typical alpha helix?
Ten amino acid residues = 1.5nm
70
What are the angles of psi and phi in alpha helix’s?
Phi = 60 degrees
Psi = 50 degrees
71
What direction do side chains face in an alpha helix?
Why?
Outwards
- inwards would cause steric hinderance and electrostatic interactions
- Maximises hydrophobic and hydrophilic reactions
- hydrogen bonds keep the helix stable
72
What do amino acid residues in an alpha helix do?
Determines the properties and how it interacts with protein domains
73
What do alpha helixes drawn as a helical wheel show?
Spatial distribution of side chains in an alpha helix
74
What shape do polypeptides in a beta sheet form?
Flat, extended structure (zig zag) that is free from steric clashes
75
What is the vertical rise per residue for beta sheets?
0.33nm
76
What is the ‘pitch’ (rise per turn) for beta sheets?
0.66nm
77
What is the typical length of a beta strand?
4-20 amino acid residues
78
What is the average length of a beta strand?
8 amino acid residues (2nm)
79
Phi and psi for a beta sheet?
Phi = -119 degrees
Psi = +132 degrees
80
How are beta sheets formed form beta strands?
- H bonds occur between N-H
81
What are adjacent, anti parallel strands connected by in beta sheets?
Hairpin turns or loops
82
What do turns / loops do in beta sheets?
Permit the change of direction of peptide chains
83
What does the curve of beta sheets mean?
Can make barrel structures
84
What are the common features of turns / loops in beta sheets?
- Variable lengths
- May be stabilised by local H bonds
- Present at the surface of molecule
- Often rich in charged / polar side chains
- Often act as binding sites
85
What are the common features of turns / loops in beta sheets?
- Variable lengths
- May be stabilised by local H bonds
86
Which form of a protein is more stable?
Folded form
87
What do polypeptide chains do in an aqueous environment?
Fold spontaneously
88
Change in Gibbs free energy (delta G) = ?
Delta G = delta H -T delta S
Delta H = change in enthalpy (heat)
Delta S = change in entropy (disorder)
T - temperature
89
Does the change inGibbs free energy need to be to positive or negative occur spontaneously?
Negative
90
What is enthalpy associated with?
The internal energy change due to the formation of non-covalent bonds such as H bonds and Van der waals interactions
91
What is conformation entropy?
A measure of the degree of conformational freedom available to a polypeptide chain
92
What happens if the hydrophobic groups are folded towards the core (away from water)?
Entropy of the water molecules in the surrounding increases
93
What do hydrophobic groups lead to in polypeptides?
A decrease in the number of H bonds that nearby water molecules can form with each other, meaning lower entropy
94
What is the hydrophobic effect of polypeptides? What does it promote?
Increased ordering (decreased entropy) due to the packing of side chains in protein ‘core’ is partially offset by decreased ordering of surrounding water molecules
-Drives the formation of hydrophobic core
95
What does increasing the entropy of water molecules do?
Provides the driving force for the hydrophobic effect?
96
What does the hydrophobic effect promote?
Tertiary structure formation
97
What can disrupt a folded polypeptide chain?
What will happen?
Is it reversible?
- High temperature
- Extreme pH
- Detergents etc.
Protein will unfold and denature
If denaturants are removed, the polypeptides can refold
98
What do folding pathways do?
Bypass random sampling of all possible conformations
99
What are chaperones?
Proteins that enhance the rate at which other proteins fold
100
How do chaperones assist folding?
- Catalyse the cis/trans isomerisation of X pro bonds
- Catalyse the interchange of disulphide bonds
- Acting as components in which single polypeptide can fold, isolated from other polypeptide molecules
101
What are neurodegenerative diseases typically caused by?
Amyloid plaques (misfolding of prion proteins)
102
What are domains?
Bundles of secondary structures that are able to fold independently of the remainder of the protein
103
What are features within a domain?
- Hydrophilic groups are exposed
- hydrophobic groups are buried (the side chains form a hydrophobic core which is closely packed)
104
What are the three classes of domain structures?
1- Alpha helical domains
2- Beta sheet domains
3- Alpha / beta domains
105
What does an alpha helical domain look like?
Pair of helixes, connected by a short loo, packed together in an anti parallel arrangement
106
How does the helixes in an alpha helical domain fit together?
Ridges of one helix fit into the grooves of its partner, creating a large complementary surface and numerous Van der waals contacts
107
What is a beta sandwich?
- Two beta sheets packed together
- Sandwich stabilised by the filling - hydrophobic side chains
- Beta sheets are often twisted
108
What is a beta barrel?
- 8 beta strands that forms a barrel like structure
- Usually has hydrophobic residues orientated towards the centre of the barrel (for proteins in an aqueous environment)
109
What is an alpha helical domain/ beta barrel?
Parallel strands for a closed beta barrel and alpha helices are located on outside only
110
What is an open twisted beta sheet?
?
Alpha helices pack against both sides of sheet
Alpha beta repeats
111
What is quaternary structure?
When more than one polypeptide chains fold together
Can be the same polypeptide (homo) or different (hetero)
112
What does only 20 amino acids mean?
Limited number meaning they cannot form all tasks required - they have to ‘recruit’ other types of chemistry
113
What is innate immunity?
Rapid but non-specific
Evolutionarily conserved
114
What is adaptive immunity?
Slow but specific
Immunological memory
115
What is immunoglobulin (Ig) / antibody
Y shaped globular proteins, produced by b cells
116
What are the 5 types of antibody?
IgA
IgD
IgE
IgG
IgM
117
What do antibodies recognise?
Unique epitope or antigenic determinant on an antigen
118
What does an antigen do?
Stimulates production of antibodies and is a target for an antibody
Recognise and bind to invaders, but not the host
119
What do Immunoglobulins bind to?
Epitopes through complementary surfaces
120
How does antibodies specify?
With their antigen binding site
121
What does immunological memory depend on?
Lymphocyte differentiation and clonal expansion
122
Process of lymphocyte differentiation and clonal expansion
1- proliferation and B cell diversification in bone marrow
2- antigen binding to specific B cell in peripheral lymphoid organ
3- proliferation (clonal expansion) and differentiator
123
How does C1 work? What does it do?
1- Binding of Ig to antigen stimulates binding of C1
2- C1 triggers amplification cascade
3- 1000’s of molecules of membrane attack complex formed
—> these punch holes in membranes
124
How does histamine release occur? (Hay fever example)
- IgE cells bind to receptors on mast cell
- multivalent antigen cross links adjacent IgE molecules
- Histamine release by phagocytosis
125
What must an immunoglobulin process?
Constant region (Fc) - that is recognised by complement C1 and by receptors (Fc receptors)
Variable domains - able to recognise an almost infinite range of antigens
Multivalency - form immune complexes
Flexibility - adjust to different spacing of antigenic determinants
126
Description of an antibody
Each antibody molecule is made of two identical light chains, and two identical heavy chains, so the two antigen binding sites are identical
127
What is the ‘anatomy’ of an immunoglobulin protein?
2 heavy chains
2 light chains
Linked by disulphides
2 arms
Symmetrical
Arms are flexible
128
How many residues does a heavy and light chain have?
Heavy - 446 residues
Light - 219 residues
129
How is the sequence divided in a heavy and light chain?
Heavy - 4 equal domains (3 are similar)
Light - 2 equal domains
130
How many c terminals are the same in heavy and light chains?
Heavy - 337 residues Light
Light - 110
131
What is the X ray structure of a constant domain?
- Beta sandwich
- 4 beta strands of anti parallel beta sheet form one fae and 3 strands from other
- domains are liked together end to end - hinge between CH1 and CH2
132
What is the x ray structure of a variable domain?
- beta sandwich but has 9 beta strands
- 4 beta strands form one face and 5 form the other
- hyper variable regions all located in loops between beta strands
133
How does each IgG have a distinct surface available for binding?
- 6 variable regions in sequence
-10^9 species of IgG, each with different HVR’s
134
What can the surface be on an IgG?
- flattish: suitable for binding to another protein
- a cleft for binding a small molecule
135
What forces stabilise the complex between Ab and Ag?
Van der Waals
Ionic interactions
The burial of hydrophobic surfaces
Hydrogen bonds
-complementary binding ensures the binding is plentiful and tight - bonds are weak on there own
136
What is the structure of the variable region bound to a lysozyme?
- classical crystal structure of complex between one arm of Ab and lysozyme
- 17 amino acid residues from Ab make contact with a total of 16 lysozyme residues
—> combined surface area = 1000A
- all six hyper variable regions contribute to the Ab-Ag formation
137
Where is hyper variable regions?
Ends of the ‘arms’
138
How are antibodies used as molecular flags?(microscope detection)
1- specific antigens are coupled with dye / gold / tag
dye = fluorescent microscope
Gold = electron microscope
139
How are antibodies used as molecular flags? (Biochemical detection)
1- antigen A separated from other molecules by electrophoresis
2- incubation with the labeled antibodies that bind to antigen A allows the position of the antigen to be determined
3- second antibody binds the first antibody
140
How do you use antibodies to purify molecules?
Immunoprecipitation and immuno affinity column chromotography
141
How does immunoprecipitation work?
1- add specific antigens-A antibodies to the mixture of molecules
2- collect aggregate of A molecules and anti-A by centrifugation
142
How does immuno affinity column chromatography work?
1- beads coated with anti-A antibodies
2- column packed with the beads
3- mixture of molecules through the column - anti a molecules bind to beads
4- elute antigen A from beads to get pure antigen A
143
What is meant by protein purification?
Techniques tailored to isolate and analyse specific proteins
Starts containing many different protein and ends in a sample containing one type of protein
144
How to purify a protein?
1- grow / obtain cells (ideally abundant in protein)
2- lose cells to release protein. Mechanical - homogenisation, sonication, high pressure disruption. Non-mechanical - detergents, organic solvents, freeze thaw cycles
3- centrifuge to remove cell debris- soluble and insoluble
4- fractionation methods
Chromatography methods
145
How to purify a protein based of properties of the proteins?
Solubility - salt fractionation
Charge - ion exchange, isoelectric focussing
Size / shape - centrifugation, size exclusion chromatography
Ability to bind various other molecules - affinity chromatography
146
What is the general principle of differential fractionation?
Reduce solubility of the protein by changing the properties of the solution (eg ph, temperature, ionic strength)
147
Disadvantage of salt fractionation?
Unlikely to get rid of all the unwanted molecules / proteins
Makes it a multi step process
148
How does column chromatography work?
1- proteins separate as they pass through the column
2- solvent continuously applied to the top of the column
Fractionated molecules eluded and collected
149
What is ion exchange chromatography
Protein has a net charge allowing it to interact with opposite charge
150
What is size exclusion chromatography / gel filtration?
Protein size in a native state. May be a multimer
151
What is affinity chromatography?
Binding of ligands
Protein specifically recognises molecule
152
What happens when you separate a protein using heat?
Solution is heated
Protein comes out of solution as it is heat labile
-protein of interest must be heat stable for this
153
How does ion exchange chromatography work?
- Positively charged protein binds to negatively charged bead
- Negatively charged protein flows through
- Increase salt concentration or change buffer pH to elute the bound protein. Gradient allows more separation
154
What is 1 band on a gel?
1 linear polypeptide chain
155
How are proteins separated by polyacrylamide gel electrophoresis?
-Sodium dodecyl sulphate (SDS) - detergent added to protein in natural state
-Protein denatured by SDS
-Reducing agents break disulphide bonds (less structural support)
-SDS gives negative charge. Moves through gel only based on size
156
How do you know what gel is in fraction?
Chromatogram using absorbance
157
What are the purification steps on the gel?
Lane M - molecular weight marker
Lanes 1- 4 - purification steps before chromatography columns
Lanes 5 - 6 - after affinity column
Lane 7 - after size exclusion column (final purification product)
158
What does gel electrophoresis do?
Separates proteins based on their size and charge
159
What does the surface charge of a protein depend on?
pH
160
What does it mean if:
PH << pI
pH = pI
pH >> pI
PH << pI protein positively charged
pH = pI no net charge
pH >> pI protein negatively charged
161
What does the charge of amino acid side change depend on?
pH
162
How doe size exclusion chromatography work?
- Carbohydrate polymer beads - small molecules Exeter the aqueous spaces within beads
- Large molecules cannot enter the beads
163
What is a native PAGE?
Protein not denatured (no SDS) and disulphide bridges not reduced (no reducing agents)
Protein retains its tertiary and quaternary structure
164
What is a homotrimer?
3 identical subunits (polypeptide chains)
165
What is isoelectric focusing?
Proteins separated based on their pI - pH at which proteins net surface charge is 0
166
What happens to soluble proteins in analytical ultracentrifugation?
Due to their thermal (brownian) motion which keeps them evenly distribute throughout the solution
167
What does light do with proteins?
Light scatters from different parts of the protein will have different intensities
This is due to constructive and destructive interference of the scattered light
Intensity of scattered light will vary with angle 0
168
What happens to light when put through a sample?
Interaction with sample results in a change to emitted light which we measure
169
How to calculate energy in protein absorption spectroscopy?
E =hv
E = energy
h = Plancks constant 6.626 x 10^-34
v = frequency (c / lambda)
170
How to calculate absorbance?
A = E cl
l = light path length (corvette width) = 1cm
c = protein concentration (unknown)
E =molar extinction coefficient of a protein
171
What do myoglobin and haemoglobin have in common?
Similar protein folds
172
What does the affect on affinity depend on?
Coordinating histidine that allows the Fe2+ to fit into the ring
173
What does myoglobin and oxygen do?
Myoglobin binds molecular oxygen tightly and releases little under physiological conditions
174
What does the His residue movement do?
Tracks movement of a helix, which bind to a dimer interface
175
How does haemoglobin achieve cooperativity?
By having two conformational states differing in affinity for oxygen
176
What does the concerted modal show?
Two states of the protein are in equilibrium, and that O2 binding stabilises one over the other
177
What is the T state and R state?
T state: Lower affinity for oxygen - deoxyhaemoglobin
R state: higher affinity - oxyhaemoglobin
178
What is the Bohr effect?
179
What does chrymotrypsin cleave after?
Aromatic or long non-polar side chain
180
What does trypsin cleave after?
Arginine, lysine
181
What defines the peptide that can be bound?
The residues lining the binding pocket?
182
What are proteases?
Enzymes that catalyse peptide bond hydrolysis
183
What are the steps of two dimensional electrophoresis?
1- Separation of proteins by pI value
2- Soaking the gel in SDS solution and fitting it on an SDS PA gel
3- Separating the proteins by molecular mass with SDS page
184
How are peptides analysed?
Mass spectrometry
185
How are proteins identified?
Peptide fragment ion spectra are matched to theoretical databases to identify the peptides which are the used to identify the protein
186
Steps for the catalysis of DIPF
1 - covalent bond between serine and enzyme
2- removal… enzyme is slowed down until free enzyme is available
3- catalytic triad makes Ser195 more reactive
4- attack by Ser195 forms a tetrahedral intermediate (stabilised by oxyanion hole)
187
What does a serine protease do?
Uses serine hydroxyl group as the attacking group
188
What happens when you smoke to cause emphysema?
- Neutrophils secrete elastase to cross elastin in the lungs
- Elastase is inhibited outside the lungs
Smoking oxidises the inhibitor
