Chapter 1 Flashcards
0
Q
What is FRET?
A
Fluorescence resonance energy transfer
1
Q
What is fluorescence microscopy used for?
A
Visualisation of interactions in living cells
Can also view co-localisation
2
Q
What can close proximity also be achieved by?
A
A common partner, not necessarily direct interactions
3
Q
What does co-immunoprecipitatation show?
A
That two molecules interact
4
Q
How do you capture the protein in co-immunoprecipitation?
A
A/G Sepharose gel
5
Q
What does the dissociation constant show?
A
The stability of the complex
6
Q
What happens if the dissociation constant is too quick?
A
Pull down assays will not work
7
Q
What does the equilibrium dissociation constant relate to?
A
Strength of interactions
8
Q
The smaller the Kd the …
A
More negative the deltaG association
9
Q
What are the limitations of the ligand binding assay?
A
Stability of complex during washing
Detection and separation may be difficult
Non specific binding
Limited to low receptor concentration
10
Q
What shape is the saturation curve?
A
Hyperbola
11
Q
What does a competition assay allow the comparison of?
A
Many ligands using the same assay format
12
Q
When May a competition assay not work?
A
When the Kc is lower than the Kd as the sensitivity is too low
13
Q
What does isothermal calorimeters measure?
A
DeltaH directly from heat released or absorbed
14
Q
Describe isothermal calorimeters
A
Label free technique
Can measure very low Kds from competition (can’t measure them directly)
Need relatively high concentrations of ligand and receptor
Ligand does not have to be small it can be large
15
Q
What can you determine in ITC?
A
Enthalpy
Entropy
Reaction stoichiometry
Kd
16
Q
To what accuracy using MS are proteins and biomolecules measured?
A
0.01%
17
Q
To what accuracy are peptides and small molecules measure to?
A
5ppm (very accurate)
18
Q
What are the three main sections of MS?
A
Ionisation source
Analyser
Detecter
19
Q
What are the two forms of ionisation?
A
MALDI: matrix assisted laser desorption ionisation
Electro spray ionisation
20
Q
Describe electrospray ionisation.
A
The sample can be directly injected into the ionisation source of the MS or the MS can be coupled to a high pressure liquid chromatography column to the sample is separated into a series of components that enter the mass spec sequentially
21
Q
Between MALDI and electrospray ionisation which can add more than one proton to high Mr’s?
A
Electrospray ionisation
22
Q
In MALDI what is the laser shone on to?
A
The crystalline solid
23
Q
Is the analyser under high pressure/vacuum?
A
Yes
24
What does the analyser separate based on?
Mass to charge ratio
25
Give two types of MS analyser.
Time of flight analyser
| Quadruple analyser
26
What does the Detecter do?
Amplifies the ion current from the analyser
27
What is the equation used to determine m/z?
((M+(nx1))/n
28
What does tandem MS show?
Structural information
29
What type of analyser is required for tandem MS?
A multi analyser eg Q-Tof, Q-Q, Tof-Tof
30
In MS/MS what is present in the collision cell?
Argon gas: inert, no reaction just collisions
31
What are the three different types of bond which can fragment along an amino acid backbone?
NH-CH
CO-NH (most common)
CO-CH
32
If trypsin cleaved at arginine what would the fragment m/z be?
175
33
If trypsin cleaved at lysine what would the m/z be?
147
34
What is the m/z reading for phosphate group?
79
35
Describe the steps of proteomics.
1) 2D gel
2) Excise protein from the gel
3) Digest with trypsin
4) Search database, MS to get masses
5) MS/MS: peptide sequences
6) Protein identification
36
What are non-covalent interactions dependent on?
pH
Temperature
Ionic strength
37
What is the reaction order definition?
The number of molecules needed to collide and from the activated complex
38
What is the equation for the first order reaction?
K=ln2/half life
39
In a mixed second order reaction what happens when B is kept constant?
The reaction is pseudo first order reaction in A
40
What type of reaction order is ligand binding?
Second order with pseudo first order conditions
41
What does the resonance in surface plasmon resonance depend on?
The Mr: when a bound receptor binds to a ligand the Mr increases and hence a different deflection and resonance occurs
42
When you measure the deflection in SPR what do you generate?
A sensogram
43
What does mass transport do?
Slows the rate of apparent kinetics
| The rate depends on the flow rate
44
What yields Kd?
The steady state
45
What factors lead to rate enhancement in enzymes?
```
Nucleophilic catalysis
Orientation and proximity
Acid base catalysis
Strain
Cofactors
```
46
Which type of reaction proceeds quicker: intramolecular or intermolecular?
Intramolecular as the reactants are already in close proximity and therefore their effective concentration is greater
47
What is the Iimited pH range for acid/base catalysis?
5-9
48
What is the pKa range for amino acid side chains able to participate in acid base catalysis?
4-10 however this can be extended by the environment
49
In acid catalysis is the pKa greater or lower than the pH?
Greater
50
What happens in general acid catalysis?
The substrate is protonated by a catalytic residue
51
What happens in general base catalysis?
The substrate is de protonated by a catalytic residue
52
What does strain do?
Causes the substrate to better resemble the transition state structure so that it does not need to undergo unfavourable structural changes during the catalytic steps
53
What metal ion cofactors have a single oxidation state?
Magnesium, calcium and zinc
Small ionic radii
Divalent
Highly polarising
54
What metal ion cofactors have a single oxidation state?
Mn, Fe, Ni and Cu
| They can all gain/lose electrons
55
What are the organic co-factors?
```
Vitamins
TPP
FMN and FAD
NAD
CoA
PLP
Biotin
```
56
What is the transition state?
The highest energy state and the least populated state
57
What is the rate determining step in enzyme reactions?
Decomposition rate of the transition state
58
What happens to the reaction rate as the energy barrier gets lower?
The rate increases exponentially
59
When is a reaction catalysed?
When the deltaG(catalysed) is less than the deltaG(uncatalysed)
60
What must happen for catalysis to occur?
The enzyme binds the transition state more tightly than it binds the substrate
61
What is an example of ordered sequential bi bi reactions?
NADH linked dehydrogenases
62
What are the two mechanisms for a ternary complex?
Ordered sequential bi bi and random mechanism
63
What are the two none ternary mechanisms?
Bi bi ping pong and theorell chance
64
Give an example of bi bi ping pong
PLP a dependent transaminases
65
Give an example of theorell chance mechanisms
Horse lover alcohol dehydrogenase
66
What does the slope graph of the ordered sequential bi bi tell you?
Y=Ka/Vmax
| Slope=Ka'Kb/Vmax
67
What does the intercept graph of both ordered sequential bi bi and bi bi ping ping tell you?
Y=1/Vmax
| Slope=Kbps/Vmax
68
What do partial reactions for bi bi ping pong show?
That it is a bi bi as only one product is produced
69
What is rule one in product inhibition pattern?
The ordinate intercept will change if the enzymes are different
70
What does rule 2 of the product inhibition pattern state?
The slope will change if the enzymes are the same or connected by reversible steps
71
Which dehydrogenases are A specific?
Malate
Lactate
Alcohol
72
Which dehydrogenases are B specific?
Glutamate
| Glyceryl delude-3-phosphate
73
What is the inhibitor of Pyruvate?
Oxamate
74
What is the inhibitor of lactate?
Oxalate
75
What are the two domains of lactate dehydrogenase?
N-terminal domain and C-terminal domain
76
What is the N terminal domain also known as? And what does it bind?
Rossmann fold and is a dinucleotide binding domain
77
What does the C-terminal bind and what is it composed of?
Substrate
| 2 x 3 anti parallel sheets
78
What is the N-terminal domain composed of?
6 stranded beta sheet
| 4 alpha helices
79
What is the composition of the Rossmann fold?
(Beta alpha beta alpha beta)^2
80
How was the rate of loop closure determined?
Site directed mutagenesis, add a fluorescent tryptophan into the loop at amino acid number 106.
The amount of fluorescent changes depending on whether it is In solvent or not, and therefore this can be studied.
Loop closure in lactate dehydrogenase is the rate determining step
81
What is the specificity constant defined as?
Kcat/Km
82
When changing the specificity of an active site what would you look at?
Overall charge balance in active site
Substrate vs active site volumes
Direct electrostatic complementarity
83
When changing LDH specificity to malate how would you change some of the charges?
Alter the amino acids in the active site to get the ideal charge of 0
84
What is glutathione reductase specific for?
NADH (catabolic) or NADPH (anabolic)
85
What family of enzymes is glutathione reductase part of?
Flavoprotein disulphide oxidoreductase
86
What residue is conserved in NADPH reactions?
Arginines
87
What is the NADPH binding consensus sequence?
GxGxxAxxxA
88
What is the binding consensus sequence for NADH?
GxGxxGxxxG
89
How many subunits are in LDH?
4: tetrameric and there are two subunit types (alpha and beta)
There are five different conformations of the subunits
90
What type of conformation of LDH is found in heart muscle?
Alpha4
91
What type of conformation of LDH is found in skeletal muscle?
Beta4
92
What mechanism does LDH a follow?
Ordered sequential bi bi
93
What did affinity labels in LDH a discover?
Residue His195
By altering Pyruvate to bromopyruvate it enabled nucleophilic attack and inactivation within the active site and as a consequences labelling of a specific residue (His195). When the enzyme was broken down, sequences and purified it could determine the specific residue.
94
What chemical modifies histidines?
DEPC
If you treat the enzyme with DEPC and you discover inactivation of he enzyme you know that a histidine is present in the active site.
95
Which chemical modifies arginine residues?
Phenylglyoxal
As you can't break down the protein and purify to a specific reside you look at the effect of activity with just the enzyme, the enzyme and substrates and phenylglyoxal and finally just phenylglyoxal. This showed substrate protection
96
What chemical modifies cysteines?
NEM
showed substrate protection also
HOWEVER be careful
Cysteine was actually in the loop and when the loop is open NEM can modify the cysteine preventing the substrate from getting in however if you add the substrate first the NEM can't then access the cysteine residue
97
What does bidirectional BLAST show?
Orthologs
98
What is BLAST bad at detecting?
Distant homologs
99
What is the order of enzymes?
OTHLIL
100
What does BLAST do?
Annotates genomes
101
What are PRIAM sequence profiles?
Multiple alignments of protein sequences
102
What is the malaria parasite?
Half plant and half animal
103
What does SHARK hunt do?
Annotation of raw genome sequence
104
In molecular networks what do the vertices represent?
Proteins
105
In molecular networks what do the edges represent?
The physical interaction between proteins
106
What are the macronutrients?
Carbohydrates, protein and fat
107
What are the micronutrients?
Vitamins and minerals
108
What is the reference nutrient intake?
The amount of nutrient needed to meet the needs of 97.5% of the population
109
What type of compound are vitamins?
Organic
110
What type of compound are minerals?
Inorganic
111
What are the fat soluble vitamins?
E, K, A and D
112
What is vitamin B1?
Thiamine (TPP)
- required in Pyruvate dehydrogenase and alpha ketoglutarate dehydrogenase
- in Pyruvate dehydrogenase nucleophilic attack occurs by the acidic carbon onto Pyruvate causing attachment of Pyruvate to the thiazolium ring
113
What does a deficiency in thiamin cause?
Beri beri
- muscles are weak caused by nerve damage
- the heart can be affected
114
What is vitamin B2?
Riboflavin which is found in FMN and FAD
| Deficiency is rare
115
What is vitamin B3?
Niacin
Component of NAD(P)H
Can be synthesised in the body from tryptophan
Deficiency leads to pellagra which has the four D's as symptoms
Rare in the developed world however it is sometimes seen in anorexia sufferers
116
What is vitamin B5?
Component of coenzyme A
117
What is the role of biotin?
It is used in carboxylases: acetyl CoA carboxylase and Pyruvate carboxylase
It is a carrier for CO2
Intracellular biotin is converted by holo carboxylase synthetase into either carboxylase biotinylation or histone biotinylation
Biocytin is converted back to intracellular biotin by biotinidase
118
What is vitamin B6?
Pyridoxane (PLP)
Used in transaminases and glycogen phosphorylase
Other roles include: haem synthesis, NT synthesis, modulates the actions of steroid hormones, inverse relationship between B6 levels and risk of cancer
119
What ion does vitamin B12 contain?
Cobalt (III) ion
Large and complex
Two enzymes which use this vitamin are L-methylmalonyl-CoA-mutase and methionine synthase
Only found in food sources
120
What caused pernicious anaemia?
Lack of vitamin B12 or an inability to process B12
121
What can pernicious anaemia lead to?
Neurological symptoms (due to myelin not being produced properly) such as an altered gait and tingling of fingers and toes or macrocytic anaemia
122
What part of folic acid is responsible for biological activity?
The pteridine ring
123
What is folic acid key in?
One carbon transfer
| Vital for DNA synthesis
124
What are one carbon transfers needed for?
The synthesis of: methionine, serine, glycine, choline, purine nucleotides and dTMP
125
What enzyme converts N5-N10 THF into DHF?
Thymidylate synthase
126
What enzyme converts DHF into THF?
Dihydrofolate reductase
127
What enzyme converts THF into N5-N10 THF?
Serine hydroxymethyl transferase
128
What can folate deficiency cause?
Megaloblastic anaemia: large immature erythrocytes
| Neural tube defects
129
What is needed for the recycling of folate?
B12
130
What vitamins are linked in DNAI methylation?
Folate, B12 and B6
131
What is vitamin C also known as?
Ascorbic acid
132
What is vitamin C a coenzyme for?
Proyly hydrolase (hydrogen bond formation) and lysyly hydroxylase (attachment sites for sugar residues, cross linking)
Bile synthesis and adrenalin synthesis
Antioxidant properties
Role in regeneration of reduced form of vitamin E
133
What can vitamin C deficiency lead to?
Scurvy
| Abnormally weak collagen
134
What is the main role of vitamin E?
Protects from free radicals and against lipid peroxidation
| Protects the nervous system
135
What is vitamin A derived from?
Dietary beta carotene
136
What is vitamin A also known as?
Retinol
137
What is the role of vitamin A?
Antioxidant and involved in sight (all trans retinol initially enters the epithelial cells and then the photoreceptor cells ... cis-retinal combines with opsin to make rhodopsin)
138
What is retionoic acid involved in?
Gene regulation
| Receptors for vitamin D and thyroid hormone which are involved in growth and differentiation of cells
139
What is the main role of vitamin K?
Koagulation
Vitamin K dependent carboxylases convert glutamate into gamma-carboxyglutamate residues which are found in clotting factors
140
What enzymes does warfarin inhibit?
Quinone reductase
| Vitamin-K-epoxidase reductase
141
What is the precursor of vitamin D?
Cholesterol
142
What does a lack of vitamin D cause?
Bone disease
Autoimmune diseases
Cancer
143
What is vitamin D a transcriptional regulator of?
Calcium homeostasis and bone growth
| Causes increase in calcium and phosphate absorption from the intestine
144
What are the roles of iron?
Electron carriers
| Component of haem
145
What does iron deficiency cause?
Microcytic anaemia
146
What caused muscle cramps and spasms?
Hypocalcaemia
147
What are plasma calcium levels controlled by?
Vitamin D and parathyroid hormone
| Stimulated when calcium levels drop
148
What is the role of iodine?
Synthesis of thyroid hormone (basal metabolic growth and essential for normal growth)
149
What does iodine deficiency cause?
```
Growth and mental retardation
Thyroid enlargement (goitre)
```
150
What controls iron absorption?
Hercipidin
151
What type of wave are X-Rays?
Electromagnetic waves
152
What happens to an electron when hit by X-Rays?
Start to vibrate at the same frequency
| Secondary beams will be scattered
153
What is the scattering from a molecule dependent on?
The number of and distances between electrons
154
What do we need in order to determine the structure of a molecule?
Amplitude and phases
155
What are the three different ways of crystal growth?
Sandwich drop
Sitting drop
Hanging drop
156
What is a unit cell?
The minimum unit able to translocated in a 2D array
157
What is an asymmetric unit?
Rotatable, smaller structure in 3D
158
What must the packing symmetry be?
Less than 6 p, but it cannot be 5 fold
159
What does Braggs law state?
Scattered beams in phase will add up and those out of phase will cancel each other out
160
Why do you get a diffraction pattern produced in spots from an X-Ray?
No lens can focus an X-Ray
161
How are the diffraction patterns interpreted?
Using the Fourier transform
162
In diffraction patterns where is the best resolution?
The further out you go
163
What is lost with the detector in X-ray crystallography?
Phases
| For small molecules you can determine them from the amplitudes however for big molecules you need another method
164
What are the phasing techniques?
Multiple isomorphous replacement
Molecular replacement
Single wavelength anomalous dispersion
Multi wavelength anomalous dispersion
165
What happens in isomorphous replacement?
You soak an atom to form a heavy atom and introduce into the crystal structure
You then look at how the diffraction pattern have altered and determine the positions of the heavy atoms and from them the phases
166
What does the intercept in MIR give you?
The phase angle
167
In MAD what happens?
You get more than one diffraction pattern and you compare them
168
What happens in molecular replacement?
Only applicable if a similar structure already exists
| You combine the phases of the known structure with intensities of unknown structure
169
What are phases?
They are the thing which contributes most to the electron density map
Contain the most important information
170
What is the R factor?
Difference between known and unknown structure
171
What is the atomic B factor?
Measure of the average displacement of an atom
172
What is the structure F factor?
Intensities from the diffraction pattern
173
What undergoes small vibrational and conformational changes in a protein?
Atoms
174
What undergoes large movements relative to each other upon ligand binding?
Large multi domain proteins
175
Can proteins undergo transient but complete unfolding? If yes, give an example.
Yes
| When oxygen is released from oxyhemoglobin
176
Can both external and internal residues be quenched?
Yes
177
What is the rate Ki?
The rate for unstructured tripeptide
178
What is rate Ko?
Rate of opening of a region of protein
179
What is rate Kc?
Rate of closing
180
What solvent are proteins put into during hydrogen exchange?
D2O
181
How can hydrogen exchange be observed by NMR?
A deuteron has a different magnetic resonance frequency to a proton and therefore as a proton is exchanged you will see the signal disappear
182
What does the rate of HX depend on?
pH
Location of the amide and degree of burying
Hydrogen bonds
Frequency of partial unfolding
183
When the protein is very big what must you combine hydrogen exchanged with?
```
2D NMR: this reduced overlapping signals
Isoelectric focussing (pI) and SDS-PAGE (mass)
```
184
How are overlapping hydrogen resonances of the amide bond resolved?
Measuring the chemical shift of nitrogen on the same amide bond
HSQC medium
This generates a fingerprint of all NH groups
185
Which residues are able to rotate giving an averaged spectra?
Tyrosine and phe
186
Which residues are unable to rotate as they are too big and therefore are classed as different hydrogens and give two peaks?
Histidine and trp
187
How do you obtain 3D structures from NMR?
Measure through space interactions/distances between NMR active nuclei. These are used as restraints when calculating structure
188
Do dynamic areas show low or high constraints?
Few constraints
189
What does the B factor in X-ray crystallography show?
The extent of smearing is given by the temperature factor B
190
Describe molecular dynamics.
Atoms of known structure are given a set velocity and random motion, after a short time the magnitude of force acting on each of the atoms is calculated and from that you can work out the new positions of each atom.
191
In molecular dynamics what is the force described as?
Sum of terms which includes bonded and non-bonded interactions
192
What occurs more often, atomic fluctuations or collective motions?
Atomic fluctuations
193
What are the four main classes of proteases?
Serine, cysteine, aspartic and metalloproteinases
194
Where does trypsin cleave?
C-terminal to a charged side chain
195
Where does pepsin cleave?
C-terminal to a hydrophobic side chain
196
Where does cleavage commonly occur?
In dynamic regions such as domain functions and loops, however fully folded native domains can be highly resistant to degradation
197
What are the two solutions for how to degrade a protein?
The proteolytic active sites are enclosed within a chamber and you use the energy to unfold the protein at a slower rate than intrinsic rate
198
What are the five bacterial proteases?
ClpXP/ClpAP
FeSH
HSIUV
Lon
199
Which bacterial proteases have the unfoldase and protease on the same peptide?
FeSH and Lon
200
What is the ClpXP composed of?
The ClpP is 2 stacked rings of heptamers and can bind two AAA+ proteins. Each monomer has a serine protease active site.
The ClpX/A site is a ring like hexamer that can bind each end of the ClpP, this protein has one AAA+ domain.
201
What does AAA stand for?
ATPases associated with various cellular activities
202
What do AAA+ proteins participate in?
Protein degradation
Protein disaggregation
Protein complex remodelling
Utilises ATPASES to catalyse the unfolding of the substrate proteins
203
How does ClpXP a recognise substrates?
It binds specific sequences/tags
Eg AADENYALAA which has two binding motifs
LAA is recognised by loop regions
AADENYAL is recognised by the adapted protein called SspB
204
What are the two common methods to measure the force applied to/by a biomolecules?
Atomic force microscope and laser traps
| Both measure the displacement of a spring with known stiffness
205
What is AFM good at measuring?
Mechanical forces over short distances
206
What are laser traps good at measuring?
Small forces over longer distances
207
What are the two types of mode of AFM?
Tapping mode and contact mode
208
What are the advantages and disadvantages of contact mode?
Any type of tip can be used and it is easy to do
| You can get sample damage and high lateral forces can cause displacement
209
What are the advantages and disadvantages or tapping mode?
A very sharp tip must be used however you reduce the lateral force and therefore reduce the damage
210
How can you make a blunt tip, sharper?
Place a carbon nanotube tip on the end
211
What does the surface area of a folding funnel describe?
The entropy
212
In a folding funnel, where is the most stable section?
The bottom
213
What do proteins fold from and to?
From: low enthalpy and high entropy
To: high enthalpy and low entropy
214
What factors determine whether a protein can be folded reversibly?
pH, temperature and adding/diluting the chaperones
215
What did the leviathan paradox show?
Proteins can't fold at random as it would take far too long, there must be a protein folding pathway.
216
What did ranganathan show?
That there is co-evolution and conservation
He showed that amino acids are not conserved independently
Carried out statistical coupling analysis
217
Out of the 36 amino acids in WW domain how m ay were needed to fold and define?
8
218
What do WW domains bind?
Proline rich sequences
219
What did Ptitsyn show?
He looked at partially folded states by using mild de maturing conditions to define the species as molten globule
220
How does a lysozyme refold?
Via parallel pathways with many intermediates
221
Describe the structure of the hen lysozyme.
```
Small with 139 amino acids
45 disulphide bonds
Enzyme glycosidase
Soluble, globular protein
Mixed alpha beta fold
```
222
How was folding kinetics studied?
Stopped flow methods
223
What does Far UV show?
Secondary structure information
| 190-240nm
224
What does Near UV show?
Tertiary structure information
| 240-300nm
225
Is the denatured state more or less fluorescent than the native state?
More
226
Is folding cooperative?
Yes
227
What else can contribute to far UV?
Aromatic residues - can give rise to an "over shoot" in the CD
228
In pulsed hydrogen exchange how do you stop hydrogen exchange?
Quench at a low pH
229
In pulsed hydrogen exchange what do the hydrogens start as?
Deuteron & an unfolded state
230
What did pulsed quench flow of lysozyme folding show about the alpha helices?
They fold quicker and cooperatively compared to beta
231
Describe the small proteins folding funnel.
Smooth
Intermediates are not populated
Few contacts are needed to define the native fold
232
Describe the folding funnel for large proteins.
```
Rough landscape
Intermediates are highly populated
Multiple pathways
Fold by domains
The hen lysozyme shows existence of distinct intermediates
```
233
What happens straight away once the protein starts re folding?
Hydrophobic collapse
234
What happens during hydrophobic collapse?
Exclusion of water
Short interactions
Molten globule state
235
Is folding uni molecular or bimolecular?
Uni
236
Is aggregation uni molecular or bimolecular?
Bi
237
How can you prevent aggregation occurring and favour folding over aggregation?
It is concentration dependent and therefore you can decrease the concentration of the unfolded state
238
How does expression of molecular chaperone usually come about?
Heat shock
239
What do molecular chaperones bind?
Any non-native protein
240
How do cells protect the polypeptide as soon as they can?
Ribosomal protein L23: specific chaperone binding site
TF and NAC: Chaperone the nascent chain as it leaves the ribosome
Hsp70/40: bind the nascent chain
241
How do heat shock proteins work?
They act as a buffer of protein aggregation: they reversibly bind misfolding proteins and therefore prevent aggregation
242
What does Hsp90 bind?
ATP and other proteins
243
What is Hsp90 involved in?
Raf-1 signalling pathway and steroid hormone receptor activation
Acts on protein conformation (modulated protein conformations)
244
What role of Hsp100 chaperones have?
Involved in protein disaggregation
245
What are the advantages of linking an unfoldase to a protease?
Efficient and safe: gated access to a potentially damaging protease activity
246
What is Hsp70 known as in Ecoli?
DnaK
247
What does Hsp70 work with?
Hsp40 and a nucleotide exchange factor
248
Which out of Hsp70 and Hsp40 binds ATP?
Hsp70
249
Which out of Hsp70 and Hsp40 has an affinity for non-native polypeptides?
Hsp70
250
What are Hsp70 and Hsp40 involved in?
ATP binding and release
251
Is GroEL essential for the viability of Ecoli under all growth conditions?
Yes
252
What is GroEL homologous to?
Rubisco binding protein
253
What is GroES homologous to?
Hsp60 in mitochondria
254
What is Hsp60 and what is Hsp10?
Hsp60: GroEL
Hsp10: GroES
255
Which out of GroEL and GroES has ATPase activity and has affinity for non-native proteins?
GroEL
256
What must be present in order for GroES to bind GroEL?
ATP/ADP
257
What are cyro-EM structures?
Imagining macromolecule complexes in a solution state
258
What did cyro-EM structures show about the ternary complex?
When ATP binds there is an enormous conformational change where the structure grows much taller
259
What does projection theorem do?
Relates the 2d image to the 3D object
| States that the Fourier transform of the 2D projection image is a central slice through the 3D transform
260
When is ATP binding positively cooperative?
Within a ring
261
What happens when ATP a binds GroE?
There is rotation within domains
262
How many continuous subunits do you need to GroEL to work?
4
263
What state is GroES release primed in?
Cigs
264
What state is GroES release triggered in?
Trans
265
What is type II Hsp60?
CCT
266
What is a replisome?
Assembly of enzymes and molecular motors that are involved with DNA replication
Coordinates synthesis of lagging and leading strand
267
Which direction does the polymerase move?
5' to 3'
268
What is T7 phage polymerase processivity factor?
Thioredoxin
269
What are the two modes of processivity with the helices even?
Processive replication
| Waiting for rebidding whilst loosely attacked to the helicase C-terminus
270
Which is the slowest step within the replisome?
The primate
271
What is key to coordination between the primase and helicase?
Zinc binding domain
272
What do the two magnesium ions of the RNA polymerase do?
Activation of the 3'-OH
| Brings in the incoming NTP
273
Newly added nucleotides bias the motion of the polymerase in the forward direction
What is the brownish Ratchet model?
274
What are the helicase superfamilies and which are hexameric and which are monomeric?
SF1, SF2, SF3, SF4, SF5 and SF6
1 and 2: monomeric
3,4,5 and 6: hexameric
275
What is the inchworm model?
Hand over hand mechanism
276
What did Koch postulate?
A series of descriptions for defining an organism
277
How can the natural flora become infectious?
Can gain extra virulence or gain access to deep tissues by trauma, surgery etc.
Or the patient might be immunocompromised and become an easy target of the natural flora
278
What does the natural flora do?
Provides colonisation resistance
Can contaminate species
Major source of disease
279
What are endogenous infections?
Caused by the natural flora
280
What is zoonosis?
The movement of diseases from an animal to a human
281
What are the two types of infection caused by tuberculosis?
Latent vs active
282
What bacteria can cause bacterial pneumonia?
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Staphylococcus aureus
Klebsiella pneuomniae
Streptococcus pneumoniae
Haemophilus influenzae
Pseudomonas aeruginosa
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283
What does pneumonia affect?
Oxygen transport through the alveoli
284
What are the two types of food borne disease?
Toxin mediated and infectious
285
What do the two subunits of cholera do?
Subunit B binds GM1 glycolipids and is the delivery portion
Subunit A causes ADP ribosylation of Gs unit meaning excessive cAMP is produced and ions are moved into the lumen affecting the osmotic balance
286
What causes endotoxin and septic shock?
Lipopolysaccharide
287
What are the symptoms of septic shock?
Fever
Hypertension
Coagulation
288
What is the plant cell wall made up of?
Cellulose
289
What is the bacterial cell wall made up of?
Peptidoglycan
290
What is the primary function of a cell wall?
Structural
291
What does a cell wall also do?
Act as anchoring points for components of the bacteria that interact with the environment eg MSCRAMMS
Supporting and protective mesh
292
Why is the bacterial cell wall important to us?
Role in virulence
Antigenic (recognised by the immune system)
Unique to bacteria (drug target)
Fundamental biological interest
293
What is the glycan strand composed of?
NAM and NAG
| From NAM extends stem peptides which cross link glycan strands
294
What form of amino acids are used?
D-amino acids
295
What is found in position 3 of the stem peptide?
Meso-diaminopimelate this contains two amino groups and therefore can form a peptide bridge
296
What is position 4?
It is always D-alanine
297
What replaces position 3 in S.aureus?
L-lys: also has two amino groups
298
When synthesised how many amino acids does the stem peptide contain?
5
299
What are the source of peptidoglycan precursors?
D-alanyl-D-alanine: which is an alanine racemase & D-ala-D-ala ligase
D-glutamate: glutamate racemase
300
In peptidoglycan synthesis what happens in stage 1?
UDP-MurNAC pentapeptide is made in the cytoplasm
| Involves MurA-F (C-F: ligases)
301
In peptidoglycan synthesis what happens in stage 2?
Moves to the membrane
MurY converts it into lipid 1 by swapping UDP for UCP
MurG adds NAG to lipid 1
302
In peptidoglycan synthesis what happens in stage 3?
Transglycosylation (polymerisation of the glycan strands) and transpeptidation (cross linking between glycan strands)
Transglycosylation occurs first carried out by PBPs
303
What is the difference between gram positive and gram negative?
Negative has two membranes: not good for tethering
304
What are the other constituents of gram negative?
Lipoproteins
Coordinates the cell wall and outer membrane
Gives rigidity
305
What other constituents are found in positive?
Teichoic and lipoteichoic acids
| Teichoic are bound to peptidoglycan whereas lipoteichoic are bound to the plasma membrane
306
What is the peptidoglycan substitute in archaebacteria?
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S-layer
Thick layer of proteins
Smaller than 8nm can fit through
Structural rigidity
Does contain NAM and N-acetyltalesminuronic acid
```
307
What is the peptidoglycan substitute in chylamydiae?
P-layer: large cysteine residues
| Can target PG synthesis with antibiotics
308
What type of immune system detects the peptidoglycan cell wall?
Innate immune system
By extracellular recognition
Intracellular recognition
Soluble peptidoglycan recognition molecules
309
What enzymes disrupt the cell wall?
Lysozyme: breaks the cell wall
Lysostaphin: metallopeptidase which targets the pentaglycine brige
310
What is the beta lactam ring a structural analog of?
D-alanyl-D-alanine
311
What enzyme does beta lactams bind to?
PBPs and causes inactivation
312
What does vancomycin do?
```
Prevents transglycosylation (steric hindrance)and transpeptidation
Forms hydrogen bonds with D-alanyl-D-alanine of the stem peptide
```
313
What are the mechanisms of antibacterial action?
Substrate analogues
Steric hindrance
Enzyme inactivation
Disruption or subversion
314
What agents target cell wall synthesis?
Beta lactams and glycopeptides (vancomycin and teicoplanin)
315
What drugs treat tuberculosis?
Ioniazoid and ethionamide
They block mycolic acid synthesis (both inhibit enoyl ACP reductase)
They are pro drug
316
What agents target the membrane?
Daptomycin: causes membrane depolarisation
| Polymysin B and E: leakage of cell content
317
Which agents target nucleotide metabolism?
Sulfamethoxazole and trimethoprim
Sulfa drugs: competitive inhibitors and alternate substrates for DHPs
Does or effect mammalian cells as we do not make tetrehydrofolate
Trimethoprim: blocks DHF being converted to THF
318
Which agents target DNA?
Quinolones and fluoroquinolones
| Effect DNA gyrae and topoisomerase
319
Which drugs effect transcription?
Rifampicin
Transcription inhibitor
Binds the beta subunit of prokaryotic RNAP
Interferes with initiation
320
Which agents target protein synthesis?
Fusidic acid and linezolid
| Bacteriostatic
321
What does mupirocin do?
Inhibits the formation of isoleucyl tRNA
322
What are the two types of antibiotic resistance?
Intrinsic and acquired (spontaneous mutation or horizontal gene transmission)
323
What are the mechanisms of antibacterial resistance?
```
Altered target site (mutation of target, increased quantities of target, modification of target)
Decreased uptake (efflux or reduced permeability)
Enzyme inactivation/modification (destruction or modification)
Target bypass (acquisition of alternative target)
```
324
Give an example of modification of target.
Vancomycin in enterococci: makes an alternative stem peptide which vancomycin doesn't bind as easily
325
Give an example of enzyme inactivation of modification.
Amino glycosidases: enzyme modifies the drug preventing interaction with target 16S rRNA
Beta lactams: cyclic amide bonds are hydrolysed and can't bind to target site
326
Give an example of target bypass.
Methicillin resistance in S.aureus
327
What are immunoglobulins secreted by?
B lymphocytes
328
What is the function of immunoglobulins?
Bind to antigens and mediate humoral immunity
329
What is the process called where gene fragments are shuffled around?
Somatic recombination of combinational joining
| This shuffling gives lots of different variable regions
330
What sections are joined with low fidelity to give junctional diversity?
V, D and J
331
Which region binds to the immune system components?
The constant region
332
Which region binds to epitopes?
The variable region
333
What is the structure of an antibody?
Dimer of dimers
| Two heavy chains and two light chains connected by disulphide bonds
334
Which domains mediate antigen binding?
Vh and Vl domains, the loops which project from these domains form the antigen binding sites and is where the sequence variation is concentrated.
335
What are the Vh and Vl loops known as?
Complementarity determining regions and hypervariable loops
336
What type of interactions are made between antigens and antibodies?
Reversible, weak non-covalent bonds
337
What are the five different immunoglobulin classes?
IgM, IgG, IgA, IgD, IgE
338
Which immunoglobulin is present in the primary response?
IgM
339
How can immunoglobulins act?
Neutralise antigens preventing entry into the cell
Opsinisation for uptake by phagocytosis
Activate the classical complement cascade
Antibody dependent cell mediated cytotoxicity
340
Which heavy chains have four CH domains?
IgM and IgE
341
How many antigen binding sites does IgM have?
Pentameric and therefore 10 binding sites
342
What does the J chain in IgM do?
Promotes IgM polymerisation
343
Where is IgM mainly found?
Due to its size it is mainly found in the vascular system
344
Does IgM have low of high affinity for antigens?
Low affinity but high avidity
345
What response does IgM bring about?
Classical complement pathway through neutralisation
346
Which immunoglobulin has four different isotopes?
IgG, 1-4
347
How does IgG bring about a response?
Opsonisation and activates the classical complement cascade
348
What is IgA secreted onto?
Mucosal epithelial surfaces
349
What is IgA's principal function?
Neutralise antigens and prevents infection in the first place
350
Where and how is IgA transported?
Across the epithelium into the mucus layer by binding to Ig receptors, they are then cleaved on the luminal surface releasing IgA
351
What is IgE an immune response for?
Worms and parasites and a few antigens
| Type 1 hypersensitivity reactions
352
What happens in class switching?
As the immunoglobulin matures it changes the expression
| Starts of expressing IgM and IgD and then goes on to express IgG, IgE and IgA
353
How is the vast repertoire of B cells generated?
Somatic rearrangement
Affinity maturation
Class switching
354
What are polyclonal antibodies?
More than one B cell is isolated
| Mixture of antibodies that respond to different parts of the protein
355
What are monoclonal antibodies?
Homogenous antibodies which respond to the same epitope of a protein
356
What is splicing driven by?
RNA
357
What did Cech show?
That splicing still occurs in simple cell extracts
358
What did Miller and Urey show?
That the prebiotic soup was: amino acids, biopolymers and nucleotide bases
359
What do you need in order for splicing to occur?
Unspliced RNA, a G nucleotide and divalent metal ions
360
What role do the divalent metal ions have?
Acid base catalysis
| Two metal ion catalysis
361
What is the hammerhead ribozyme?
Viroids that do not code for any gene products and are not long
They kill by continuous replication (hijack cells enzymes etc) and messing up cellular metabolism
Consist of ssRNA
362
What is the mechanism of ribonucleotide reductase?
Ribonucleotides are made first and then converted into 2'-deoxynucleotides in a highly conserved reaction
363
What is SELEX?
Systematic evolution of ligand by exponential enrichment
