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Block 2 - energy and proteins Flashcards

1
Q

what is the difference between an endergonic and exergonic reaction

A

endergonic reactions require energy whereas exergonic reactions release energy

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2
Q

what is the condition required for endergonic and exergonic reactions to be coupled

A

the energy released by the exergonic reaction needs to be greater than the energy required by the endergonic reaction

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3
Q

define gibbs free energy

A

the amount of energy available from a particular compound

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4
Q

the gibbs free energy of an endergonic reaction is negative/positive

A

positive

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5
Q

the gibbs free energy of an exergonic reaction is negative/positive

A

negative

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6
Q

what are the components of ATP

A

adenosine (adenine + ribose) + 3 phosphates

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7
Q

describe the two methods of ATP hydrolysis

A

ATP –> ADP and an inorganic phosphate
OR
ATP –> AMP and pyrophosphate. the pyrophosphate is then split into single phosphates

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8
Q

describe the energetic differences between the 2 mechanisms of ATP hydrolysis

A

the ATP –> AMP method releases more energy than the ATP –> ADP mechanism but also requires more energy to reverse

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9
Q

give 3 examples of what ATP can be used for

A

chemical work, transport and movement

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10
Q

why does the ATP turnover need to be so fast

A

we need much more ATP per day than what is stored in our body (100-250g stored - need 50-75kg). ATP needs to be turned over around 500 times per day to meet the daily requirement

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11
Q

describe the energy sources during 100m sprinting, where ATP turnover is not enough to sustain the body

A

stored ATP - 1 sec
creatine phosphate - 4 sec
fermentation of glucose - rest of race

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12
Q

which product of anaerobic metabolism can be very painful

A

lactic acid

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13
Q

high/low [ATP] inhibits catabolic reactions and stimulates anabolic reactions

A

high

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14
Q

why is it sterically difficult to attach a phosphate to ADP

A

we need to overcome the repulsive negative charges of the other phosphates
because of this a lot of energy is released when a P is released

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15
Q

give examples of reactions that require phosphate from ATP

A

FA –> phospholipid
synthesis of RNA/DNA - nucleoside monophosphate activation
protein synthesis - amino acid activation
post translational activation/deactivation by phosphorylation

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16
Q

what doe kinases do

A

they phosphorylate

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17
Q

what do phosphatases do

A

they dephosphorylate

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18
Q

describe the nitrogen sensor in plants

A

NRTI.I nitrate sensor/transporter in plants - P site in thr 101nprovides a switch between high and low affinity nitrogen uptake

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19
Q

describe calcium active transport

A

Ca pumps maintain low cytoplasmic concentration to allow fast transient signals (requires ATP)

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20
Q

what is the first internal messenger in guard cells and what does it do

A

intracellular calcium - it opens channels so that ca can be released very quickly

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21
Q

describe the myosin and actin motion in muscle and the involvement of ATP

A

myosin movement relies on reversibly binding to the actin filament and the unbinding requires ATP

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22
Q

what are the 2 ways that ATP can be synthesised

A

substrate level (P group transfer) or oxidative phosphorylation (H gradient, ATP synthase)

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23
Q

to make ATP the substrate needs to have more or less energy than ATP

A

more

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24
Q

why do p groups have high energy

A

because they are attached in different types of bondages which are energy rich

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25
Q

give an overview of the ETC

A

NADPH brings energy rich e- that have been harvested through metabolism and they go down the chain towards the final electron acceptor (O2). the energy released is harvested by a proton gradient (matrix –> intermembrane space). H flow down their concentration gradient through ATP synthase, making ATP

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26
Q

describe mitochondria

A

numerous in ATP consuming tissues and closely associated with ATP consuming organelles
contains membrane protein complexes that are important in respiration

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27
Q

describe the structure and function

A
  • rotor - spins clockwise when H flows past
  • stator - holds rotor and knob in position
  • rod - turns with rotor and causes a conformation change that activates the knob
  • knob - catalytic sites join Pi + ADP making ATP
    ATP synthase is made by separate proteins encoded by different genes
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28
Q

how does cryo EM work and why is it useful for visualising ATP

A

solubilized protein spotted on gold coated grid and freezing - then normal EM
it can be used to look at ATP in different dynamic structures

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29
Q

describe ATP synthase F1 structures

A

different Beta subunit forms have different affinities for ATP and ADP
alpha subunit rotation changes beta subunit conformation
the alpha subunit depends on shaft position for conformation - one conformation binds ATP, one makes it and one releases it
ATP drive the pump counter clockwise

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30
Q

describe the reversibility of ATP synthase

A

in low [ATP] ADP + Pi –> ATP

in high [ATP] ATP –> ADP + Pi

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31
Q

what issue could the reversible nature of ATP synthase pose if it is not controlled properly

A

we need to avoid the ATP being generated being removed again due to the reversible nature. to avoid this ATP is quickly removed after being generated

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32
Q

what does the ADP-ATP exchanger do

A

it keeps [ATP] low in the mitochondrial matrix by removing ATP and adding ADP

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33
Q

what does the Pi/H cotransporter do

A

uses energy of the H gradient to import Pi

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34
Q

what is the overall reaction in glycolysis

A

glucose –> 2x pyruvate

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35
Q

what is pyruvate transformed into which is then used in the citric acid cycle

A

acetyl coA

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36
Q

when in respiration does oxidative phosphorylation occur

A

in the ETC and chemiosmosis

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37
Q

in respiration how many ATP are produced and by what means

A

2ATP by substrate level phosphorylation

32/34ATP by oxidative phosphorylation using ATP synthase and the proton gradient

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38
Q

what do NAD FAD and NADH all have in common

A

they can all reversibly uptake electrons
they can be oxidised - no electrons
they can be reduced - with electrons

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39
Q

how does NAD+ change to form NADH

A

double bonds are altered to accommodate the electrons (2e- + H+)

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40
Q

write the equation for a basic redox reaction

A

Xe- + Y –> X +Ye-

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41
Q

what is the redox potential

A

relative affinities of atoms to their outer shell e-

the differences in the redox potential provide a source of energy

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42
Q

describe reducing sugars

A

those that want to get rid of electrons - we harvest the potential energy being released by allowing e- to move from the sugars to O2 ultimately

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43
Q

molecules that have very -ve redox potential have a lot/little free energy

A

lots

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44
Q

why are the mitochondrial membrane protein complexes important

A

they enable e- transfer and harvest energy to pump protons from the matrix into the intermembrane space which is used to drive chemiosmosis driven

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45
Q

describe the transfer of energy in 3 steps in respiration

A

redox energy –> electrochemical energy –> ATP

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46
Q

describe the electron transfer from NADH to O2

A

NADH e –> NADH reductase (first electron carrier protein complex) –> ubiquinone (Q) (first electron acceptor which now becomes reduced) –> cytochrome C (electron acceptor) (catalysed by the second protein complex - cytochrome reductase) –> O2 (catalysed by cytochrome oxidase)
energy released throughout the process is used to pump protons

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47
Q

what is the dual action of the protein pumps in the respiration ETC

A

they are redox enzymes and proton pumps

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48
Q

what are the 3 protein complexes in the respiration ETC

A

NADH reductase
cytochrome reductase
cytochrome oxidase

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49
Q

how do we measure redox potential

A

measured as voltage using voltmeter

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50
Q

what are all the redox reactions in the respiration ETC

A 
NADH  NAD+ + H+ +2e-
reduced Q  oxidised Q + 2H+ + 2e-
reduced cyt c  oxidised cyt c + e-
H2O  1/2O2 + 2H+ 2e-
this process is energetically downhill and there is progressive increase in redox potential and decrease in free energy
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51
Q

what is the energetic difference approximately between NADH and oxygen in respiration

A

over 1 V

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52
Q

what is the equation used to work out free energy using the redox potentially measured experimentally

A

deltaGo = - n(0.023)deltaEo(mv)
where n is the number of electrons involved
and Eo is the redox potential

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53
Q

how is FADH2 involved in transferring electrons to the ETC in respiration

A

it passes electrons to succinate Q reductase which then passes electrons to ubiquinone (Q)

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54
Q

is succinate Q reductase a proton pump

A

no

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55
Q

what are the prosthetic groups of the 3 protein proton pump complexes and succinate Q reductase of the ETC in the order in which they are involved in the pathway

A

NADH reductase - FMN, FeS
succinate Q reductase - FAD, FeS
cytochrome reductase - Heme (b,c), FeS
cytochrome oxidase - Heme (a), Cu

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56
Q

why do we get less energy from FAD than NAD

A

because succinate Q reductase is not a proton pump

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57
Q

describe the common redox groups

A

flavins e.g. FMN, FAD - ring structures with double bonds
quinone e.g. ubiquinone
heme group e.g. cyt c - ring structure with central Fe (5 different heme groups in the ETC
FeS clusters - either 2Fe-2S or 4Fe-4S, both have only 1e- (7 different FeS clusters in the ETC)

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58
Q

why is it useful that the redox groups are coloured

A

they can be monitored easily by looking at the colour change upon oxidising or reducing

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59
Q

what happens to complexes when ETC blockers are applied

A

they are usually in a mixed state but will become fully oxidised or reduced depending on their position relative to where the blocker acts
if they act before the blocker they will become fully reduced due to electron build up and if they act after they will become fully oxidised because electrons have been blocked from flowing to them

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60
Q

what happens to protein complexes in the ETC when oxygen is removed and why can removing O2 then adding it back be useful

A

they all become fully reduced
when O2 is added back the protein complex closest to O2 will change colour and oxidise firs because it loses its electrons

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61
Q

describe NADH reductase

A
  • a large portion of it is in the membrane
  • the alpha helices are in the membrane
  • soluble blob has FeS prosthetic groups
  • Q binds at the interface of the blob and the intermembrane part
  • NADH brings electrons to the top of the enzyme
  • it has some flavin groups
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62
Q

describe cytochrome reductase

A
  • dimer
  • intermembrane part and blob
  • heme groups
  • Q delivers 2e- but complex can only carry 1 (problem - alone they are radicals - can damage other molecules by stealing e-) radicals are held deep in the core to safeguard the environment
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63
Q

describe cytochrome oxidase

A
  • 1 e- delivered from cyt c but 4 needed for O2. e- are stored until there are 4
  • conserved structure
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64
Q

describe the Q cycle

A
  • Q gives one e- to cyt c and the other is buried in cyt c reductase by another Q molecule. the second e- pair comes along from another Q and one binds to the radical, the other electron goes to cyt c. reduced Q is released
    other explanation
  • e- transfer from Q (2e-) to cyt c (1e-). it requires a repetitive cycle in which radical intermediate is held inside the enzyme in a Q molecule buried in the structure. the next Q goes into the chain and transfers one e- to be buried and the other pairs with the previous radical.
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65
Q

where did photosynthesis originate and what did it result in

A

it originated in cyanobacteria and this lead to atmospheric oxygenation

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66
Q

in a basic explanation how did chloroplasts come about

A

early photosynthetic bacteria were engulfed by eukaryotic cells and transformed into chloroplasts

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67
Q

name 2 photosynthetic organisms only found in the top layer of the ocean

A

phytoplankton and seaweed

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68
Q

why is wild vegetation better at capturing carbon than agricultural vegetation

A

we don’t grow crops throughout the year in agriculture and we don’t exploit the whole 3D space

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69
Q

there is a loss/gain of carbon stock upon land conversion from wild to agricultural

A

loss

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70
Q

to sustain the growing population we need to intensify ………, not expand land

A

fixation

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71
Q

describe chloroplasts

A

made from membranous structures that carry photosystems
2 membranes - outer belongs to the plant, inner is of bacterial origin
thylakoids - stacks of membranes called grana. inner membrane invaginations were cut off to become thylakoids
interior - stroma
move towards light and communicate with each other

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72
Q

highlight some differences between mitochondria and chloroplasts

A
  • chloroplasts are larger
  • different sub compartmental structures
  • chloroplasts capture light to make ATP whereas mitochondria use NADH to make ATP
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73
Q

highlight some similarities between mitochondria and chloroplasts

A
  • inner membranes carry redox enzymes
  • both have ATP synthase - in the mitochondria the knob sits in the matrix and the protons accumulate in the intermembrane space. in chloroplasts the knob sits in the stroma and the protons accumulate in the thylakoids
  • they both have their own DNA but are no longer self sufficient
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74
Q

what is the overall role of photosynthesis

A

to use light to fix CO2 into organic molecules

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75
Q

what do photosynthesis and respiration have in common

A
  • they both have an ETC, redox reactions and H pumps
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76
Q

what are the two main steps of photosynthesis

A
  1. energy capture (ATP, NADPH production) - the photosystems in the thylakoids use sunlight to extract an e- from water
  2. build up of organic carbon molecules from CO2 (ATP, NADPH consumption)
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77
Q

the calvin cycle can only operate in the dark true or false

A

false
the calvin cycle can occur in the light or the dark it just so happens that it is referred to as the dark cycle because light is not a requirement

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78
Q

describe the electron flow in photosynthesis

A
  • plastoquinone –> plastocyanin (energetically downhill, energy harvested in pH gradient)
  • PS2 - light energy –> photoexcited e- –> plastoquinone (electron void in PS2 allows it to pull an electron from water)
  • PS1 - receives e- and promotes them to a higher energy state using light, e- shuttled to ferrodoxin then NADP
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79
Q

electron void in PS2 allows it to pull an electron from water. what is the opposite of the this step

A

it is the opposite to the final step of respiration

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80
Q

in respiration electrons lose free energy going uphill/downhill to oxygen

A

downhill

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81
Q

describe the z scheme

A

this is the energy scheme seen in photosynthesis
it is composed by and uphill movement followed by a downhill movement followed by another uphill movement
there is an overall increase in energy

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82
Q

describe chlorophyll

A
  • sit in the centre of PS
  • ring structure, double bonds, Mn4 allows reversible e- uptake
  • absorbs blue/red light
  • hydrophobic tails plant it in the membrane
  • there are different forms with different absorption spectra
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83
Q

what is a PS made up of

A

antenna complex - protein and chlorophyll array

reaction centre - contains special pair of chlorophyll molecules

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84
Q

what is the role of the antenna complex

A

collects and funnels energy to the reaction centre

it expands the light capture range

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85
Q

what is the role of the reaction centre

A

produces high energy electrons and passes them to quinone

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86
Q

what is decay by resonance transfer

A

it is what chlorophyll molecules do in the antenna complex

they pass on the excitement only

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87
Q

what is decay by successive electron transfer

A

it is what electrons do in the reaction centre - they pass e- to e- acceptor

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88
Q

what are the 3 types of photosystem

A

P900 - bacteria
P680 - PS2
P700 - PS1

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89
Q

how do the different types of PS differ from each other

A

the size of the antenna complex and the initial e- acceptor differ as well as the maximal absorbance

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90
Q

describe the electron flow in the purple bacteria reaction centre

A

special chlorophyll –> chlorophyll –> pheophytin –> tightly bound quinone –> free quinone (leaves PS so e- can’t drop back down (speed also prevents this))

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91
Q

describe the electron flow in the reaction centre of plants

A

light energy to P680 Mn4 –> special chlorophyll –> chlorophyll –> pheophytin (first e- acceptor) –> plastoquinone –> exchangeable plastoquinone –> (PSII–>PSI) –> plastocyanin –> chlorophyll (first e- acceptor) –> quinone –> FeS complex –> ferrodoxin
see 1st year notes for full process

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92
Q

how can the ATP : e- be changed in photosynthesis

A

by changing from the z scheme to cyclic phosphorylation to make more ATP (electrons are passed back to quinone)

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93
Q

what are the functional protein classes (7)

A 
structural 
scaffold 
enzymes 
membrane transport 
motor 
regulatory 
molecular machines
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94
Q

give an examples of a protein that belongs to several functional classes

A

insulin receptor - membrane transport, regulatory, enzyme

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95
Q

describe structural proteins

A

they determine the cell shape and contribute to the extracellular environment

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96
Q

give an example of structural proteins and their role in the cell

A

actin and tubulin - movement and shape

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97
Q

what are microfilaments made of

A

actin monomers

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98
Q

what are intermediate filaments made from

A

rope like assemblies of fibrous protein e.g. keratin

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99
Q

what are microtubules made of

A

cylinders made tubulin dimers of alpha/beta tubulin

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100
Q

describe scaffold proteins

A

they bring proteins into ordered complexes e.g. bring kinases together, formation of enzymes, molecular machines. they are often bound to adaptors to direct them to a specific point

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101
Q

describe membrane transport proteins

A

they are embedded in the membrane and recognise specific classes of molecules

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102
Q

give an example of structural proteins and their role in the cell

A

actin and tubulin - movement and shape

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103
Q

what are microfilaments made of

A

actin monomers

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104
Q

what are intermediate filaments made from

A

rope like assemblies of fibrous protein e.g. keratin

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105
Q

what are microtubules made of

A

cylinders made tubulin dimers of alpha/beta tubulin

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106
Q

describe scaffold proteins

A

they bring proteins into ordered complexes e.g. bring kinases together, formation of enzymes, molecular machines. they are often bound to adaptors to direct them to a specific point

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107
Q

describe membrane transport proteins

A

they are embedded in the membrane and recognise specific classes of molecules

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108
Q

describe regulatory proteins and give examples

A

they alter functions of other proteins e.g. receptors and signalling proteins

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109
Q

describe motor proteins and give examples

A

they move proteins, cells, organelles and organisms e.g. actin, myosin, kinesin ( head walks along microtubules, moving transport vesicles anchored to the tail - ATP dependent)

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110
Q

increased concentration increases/decreases collision frequency

A

increases

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111
Q

most collisions and successful/unsuccessful and why

A

unsuccessful - non covalent interactions are weak and transient

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112
Q

describe the 2 different types of dimerization

A

same protein dimerises

protein-ligand dimerization

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113
Q

list non covalent interactions in order of decreasing strength

A

ionic bonds
H bonds
hydrophobic interactions
van der waals

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114
Q

what is the approximate strength of a covalent bond

A

~200kJ/mol

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115
Q

more non covalent bonds –> more tightly bound –> higher/lower affinity

A

higher

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116
Q

why does the actin and myosin interaction need to be carefully balanced

A

it needs to be tight to withstand forces but loose enough to be reversed

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117
Q

higher affinity interactions require more/less energy to break

A

more

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118
Q

describe the 2 different types of dimerization

A

same protein dimerises

protein-ligand dimerization

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119
Q

what is a dialysis chamber and how is it used to study the R2C2 complex

A

it is a chamber with sides separated by a semi permeable membrane
- radioactive cAMP (can pass) is put in one side and R2C2 in the other (cant pass). we then measure the [cAMP], the lower the concentration at equilibrium the higher the affinity (keep R2C2 concentration constant.

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120
Q

what is induced fit

A

enzymes permit small latitude in structure of substrate leading to induced fit where the protein changes conformation when the ligand binds

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121
Q

describe the structure and mechanism if the cAMP dependent protein kinase

A
  • it has 2 regulatory and 2 catalytic subunits. the regulatory subunits have the cAMP binding sites and the catalytic sites have the kinase activity
  • the subunits form the R2C2 complex held together by non covalent interactions
  • R masks the kinase active site
  • when [cAMP] rises it is bound by the R subunits which leads to a conformation change (allosteric regulation) weakening the R2-C2 interaction. the C subunits dissociates and catalytic activity resumes
  • the system is dependent on binding and is reversible if cAMP is removed
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122
Q

what is Kd

A

the binding dissociation complex
[P][L]/[PL]
Koff/Kon

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123
Q

how do we measure Kd

A

we either measure the equilibrium concentrations and calculate or we measure the kinetics of dissociation/association and calculate

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124
Q

what is the relationship between Kd and affinity

A

lower Kd = higher affinity, lower [L] needed to bind 1/2 P

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125
Q

what is a dialysis chamber and how is it used to study the R2C2 complex

A

it is a chamber with sides separated by a semi permeable membrane

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126
Q

what information is important to understand binding

A

structural information

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127
Q

what is the primary structure of a protein

A

sequence of amino acids connected by peptide bonds in a polypeptide chain

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128
Q

what is the secondary structure of a proteins

A

alpha helices, beta sheets and beta turns formation mediated by H bonding in the backbone

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129
Q

what is the tertiary structure of proteins

A

folding into the 3D shape by interactions between R groups

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130
Q

what is the quaternary structure of a protein

A

multiple polypeptide chains coming together

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131
Q

how can the peptide bond be best described

A
  • as resonance structures - there are 2 resonance structures
  • polar
  • electrons are delocalised
  • rotation around the C-N bond is restricted
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132
Q

describe the alpha helix

A
  • common structural motif mediated by H bonding
  • H bonding between the N-H and the C=O of a residue 4 residues away
  • there are 3.6 residues per turn
  • always right handed
  • R groups point outwards
  • proline residues are helix breakers (disrupts helical structure
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133
Q

describe beta sheets

A
  • formed by H bonds between protein strands rather than within strands
  • R groups alternate above and below the plane of the sheet
  • amino acids are more extended than in the alpha helix
  • sheets can be parallel or anti parallel
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134
Q

most common chains can form ……. ……… and …….. ………..

A

alpha helices and beta sheets

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135
Q

describe the beta turn

A
  • they allow polypeptide chains to turn and go in the opposite direction
  • they allow proteins to attain a compact (globular) shape
  • proline glycine are commonly found in the structures
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136
Q

why are proline and glycine commonly found in the beta turn structures

A

proline because it is cyclic - 5N ring - connects to backbone twice which facilitates turns/kinks
glycine because of its small side chain - can fit wherever it is required

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137
Q

list some tertiary structure R group interactions

A 
H bonding 
ionic bonding 
van der waals 
hydrophobic interactions 
disulphide bonds
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138
Q

describe tertiary protein structure hydrophobic interactions

A

hydrophobic R groups cluster in the inside of the protein leaving hydrophilic amino acids on the outside

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139
Q

describe the tertiary structure disulphide bonds

A

covalent linkages between S containing side chains of cysteine residues
these are stronger than the other types of tertiary bonds and they bring the peptide round to fold

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140
Q

which level of protein structure contains all the folding information

A

primary structure

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141
Q

proteins fold to the lowest/highest free energy conformation

A

lowest

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142
Q

what happens to free energy and entropy as proteins folding proceeds

A

they both decrease to a minimum

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143
Q

why do linear amino acid chains have high entropy

A

because they have multiple possible conformations

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144
Q

most proteins begin to fold in ………….

A

translation

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145
Q

what is the molten globule

A

a partially folded state which conserves native-like secondary structure content without the tightly packed protein interior
it has intermediate energy and entropy

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146
Q

describe the change between native, molten globule and denatured proteins

A

native protein (slow) molten globule (fast) denatured

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147
Q

which protein state has the lowest free energy and entropy

A

the native state - fully folded

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148
Q

how are van der waals useful in proteins

A

they help atoms in a protein pack together

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149
Q

why are alpha helices and beta sheets highly conserved

A

natural selection favours protein sequences with a single conformation which forms easily and seldom makes mistakes

150
Q

describe the hydrophobic effect

A

it is the tendency of non polar molecules to aggregate in water

  • water has flickering cluster structure, it H bonds with 7 or 8 other water molecules. the hydrophobic effect is an indirect effect resulting form water H bond exchange rate being 1011s-1
  • non polar molecules result in less H bond opportunities for water and longer H bond lifetime (ice structure) –> decreased entropy
  • at the interface water is rotationally and translationally constrained
  • to minimise the effect of water on entropy, molecules that are hydrophobic cluster in the interior to minimise entropy loss
151
Q

give 2 examples of diseases that are caused by aberrant protein folding

A

mad cow disease and Alzheimer’s

152
Q

describe how aberrant protein folding leads to Alzheimer’s

A

molten globule –> self association of globules –> amyloid fibril core structure –> protofilament of partially folded amyloid proteins

153
Q

what is a domain

A

it is a conserved part of the protein structure that can evolve, function and exist independently
they are the building blocks of proteins
many proteins consist of different domains linked together

154
Q

why is the fact that domains fold independently key

A

it means that they can be easily moved between proteins by evolution or by genetic engineering

155
Q

what is the chicken sarcoma

A

it is caused by the rous sarcoma virus which contains the src viral oncogene (tyrosine kinase) which has 3 domains: SH3, SH2, and kinase

156
Q

what do SH3 domains bind to

A

polyproline motifs

157
Q

what do SH2 domains bind to

A

p-tyr residues

158
Q

describe the SH2 domain

A

structurally conserved and autonomously fold - they all exhibit the same arrangement of alpha helices and beta sheets and also have conserved regions of primary structure
they are important in signal transduction

159
Q

what is a bromodomain

A
  • a domain that binds acetylated lysine residues
  • key residues have been identified in addition to the acetylated lysine
  • they all bind slightly different acetylated lysines
  • different bromodomains control different bromodomains control different aspects o histone reading activity. although they have similar folding and homologous structures they are sufficiently different to be targeted by different inhibitors
160
Q

how is a motif different from a domain

A

it doesn’t depict a functional role although it can sometimes indicate towards function
it isn’t independently stable
domains tend to relate more to structural units

161
Q

what is a motif

A

it can often define functional characteristics and are usually predictive of belonging to particular groups
they can be used to predict families e.g. kinase motifs

162
Q

what is the walker motif

A

ATP binding site

163
Q

exon ……….. in evolution contributed to shaping of eukaryotic proteomes

A

shuffling

164
Q

what is the difference between the folding of big and small proteins

A

small proteins fold on their own but big proteins have domains which fold when synthesised and are independently stable while the rest of the protein is still being synthesised

165
Q

what do the linker regions between domains do

A

they are often unstructured and act like a flexible hinge

166
Q

why are active sites structurally difficult too determine

A

because the residues may be spread out in the primary sequence which may be close together in the folded protein

167
Q

what are in silico approaches

A

computerised approaches

168
Q

how can we use genome sequencing to predict protein structure

A

we can infer the sequence of amino acids and form this the structure

169
Q

what is a blast search

A

basic local alignment search tool

algorithm for comparison of sequence data

170
Q

what is an MSA

A

multiple sequence alignment
sequence alignment of 3 or more sequences
used to assess conservation of protein domains, secondary and tertiary and secondary structures and even individual amino acids

171
Q

……………………… analysis can assess the shared evolutionary origins

A

phylogenetic

172
Q

what are atypical protein kinase Cs

A

they are a subfamily of kinases composed of two members with 72% identity

173
Q

what does the structure of a protein provide that gives an insight into function

A

it allows an understanding of 3D arrangement and an insight into mechanism

174
Q

describe X ray crystallography

A

a form of microscopy for the visualisation of protein structure at atomic level
the visual detail is limited by the electromagnetic wavelength
electron density is used to determine the atomic conditions

175
Q

what are the steps in X ray crystallography

A

the protein first needs to be purified and crystallised then X rays are fired through the sample, producing a diffraction pattern which is interpreted by a computer and a crystallographer

176
Q

what is the problem with X ray crystallography

A

many proteins only crystallise at non-physiological pH or [salt] hence can be difficult to crystallise

177
Q

apart from S ray crystallography what are other methods used to determine protein structure

A

NMR and EM

178
Q

what information does NMR provide

A

provides structural information under more physiological conditions (in aqueous buffer)

179
Q

what information does EM provide

A

it provides the overall molecular shape but gives less of a sense of the individual atoms

180
Q

we can use ………… information in designing inhibitors/activators/targeted drugs

A

structural

181
Q

what are ACE inhibitors

A
  • angiotensin converting enzyme inhibitors
  • angiotensin usually converts angiotensin I to angiotensin II but the ACE inhibitors prevent angiotensin II production acting to lower bp
182
Q

what is meant by design of “better” drugs

A

less side effects
increased efficacy
less need for combination therapy

183
Q

what are the different methods of drug discovery

A
  • go through a chemical library and see what will act as a ligand, docking to the protein
    OR
  • reverse docking - take already existing drug and add proteins from target database. for proteins that bind it may be an effective drug for them.
184
Q

what is homology modelling

A

it is based on the observation that 2 proteins belonging to the same family with similar protein structures will have similar 3D structures. this is used frequently in the pharma industry

185
Q

the degree of 3D structure conservation in a family is much less/greater then sequence conservation

A

greater

186
Q

why do proteins in the lab need to be kept on ice and with protease inhibitors

A

proteins are susceptible to protease degradation and are also affected by other environmental conditions

187
Q

when cells are broken open what environmental factors need to be controlled for to maintain confirmation

A

[salt] and pH

188
Q

what is size exclusion chromatography

A
  • protein solution run in column of gel beads (small proteins move into channels and pores getting stuck and larger proteins will elute first)
  • it works best on water soluble proteins
  • it is easy and cheap and begins to fractionate the mixture
189
Q

why can we separate proteins based on charge

A

at the isoelectric point a protein has not net charge. at a pH above the pI a protein has a negative charge and below a positive charge
we can make the desired protein have a particular charge so it can be isolated, or at least purified more

190
Q

describe anion exchange chromatography

A

we have a positively charged resin or beads that binds negatively charged proteins

191
Q

describe cation exchange chromatography

A

we have a negatively charged resin or beads that binds positively charged proteins

192
Q

describe affinity chromatography

A

a protein mix is added to a column containing polymer bound ligand specific for the protein of interest. unwanted proteins wash through. to elute the protein of interest that has bound we need to wash the column with the ligand. this is a more specific method but we need to know the ligand

193
Q

what is isoelectric focusing gel

A

it is based on SDS page. proteins have first been separated on an ion exchange strip which separates proteins based on pI and pH gradient. each spot represents and individual protein

194
Q

what is proteomics

A

large scale analysis of proteins, in particular complex mixtures e.g. cells, organelles, viruses.

195
Q

what advances have lead to proteomics becoming a large area of study

A

it is enabled by the accumulation of DNA and protein databases, improvement of computer algorithms and mass spec

196
Q

list the transcriptome, proteome and genome in increasing order of complexity

A

genome –> transcriptome –> proteome

197
Q

what is mass spec

A

It involves measuring the mass : charge ratio of each amino acid.
small vaporised peptides are passed through magnets which causes bending. the bend extent depends on the mass : charge. the detector measures ionic collisions and readouts give the peptide sequence

198
Q

enzymes work by decreasing/increasing activation energy

A

decreasing

199
Q

do enzymes change the equilibrium of a reaction

A

no

200
Q

enzymes are usually highly specific for particular substrates true or false

A

true

201
Q

give examples of enzyme uses in industry

A
  • biological washing powder contain lipases and proteases to remove protein and FA stains respectively
  • many drinks and prepared food have high fructose corn syrup (starch is broken down by amylases and glucose –> fructose by xylose isomerase)
202
Q

what did frances Arnold do

A

she induced mutations through error prone PCR and selected enzymes with better properties

203
Q

what is the rate equation

A

rate = k[A][B] where k = the rate constant

204
Q

the half life of an enzyme is long/short if it is stable

A

long

205
Q

progress curves allow measurement of ………. ………..

A

initial rate

206
Q

what is a progress curve

A

it shows the disappearance of reactant or the appearance of product over the time course of the reaction

207
Q

how can we infer the initial rate from the progress curve

A

the initial rate is the slope of the initial line. formation of product is initially linear with time.

208
Q

initial rate is directly proportional to ………… ……………..

A

enzyme concentration

209
Q

reactions proceed via the ……….. ……………

A

transition state

210
Q

what is the transition state

A

an intermediate state where the nucleophile and the leaving group are bound

211
Q

what are the 2 requirements for a collision to be successful

A

the reactants need to have correct orientation and thermal energy

212
Q

…….…… stabilise the transition state

A

enzymes - they lower Ea

213
Q

what is a Boltzmann distribution curve

A

it shows the kinetic energy on the x axis and on the y axis the number of molecules with each kinetic energy

214
Q

what happens to the Boltzmann distribution curve when temperature of a reaction is increased

A

it shift lower and to the right

215
Q

what is the Arrhenius equation

A

k = Aexp(-Ea/RT)
r is the gas constant
k is the rate constant
T is the temperature in K

216
Q

in an lnk vs. 1/T plot what is the slope representative of

A

-Ea/R

217
Q

enzymes are usually more/less stable in the cold. with increasing temperature they usually ……………..

A

more

denature

218
Q

initial rate increases roughly ……………. for every 10 C increase in temperature

A

double

219
Q

why is optimal temperature a meaningless concept

A

because rate can go up but the enzyme might denature

220
Q

why do pepsin and trypsin have different optimal pHs

A

because they function in different places. pepsin (pH 1.5) functions in the stomach so needs to tolerate very acidic conditions but trypsin functions in the intestine which is a more neutral environment so its optimal pH is around 7

221
Q

what groups does the active site contain

A

groups that bind to the substrate in a defined orientation and groups that help catalyse the reaction

222
Q

the ………. ………… stabilises the transition state

A

active site

223
Q

enzymes are larger/smaller than their substrate

A

much larger

224
Q

…….……. ………….. can lead to different enzymes with similar mechanisms

A

convergent evolution

225
Q

give an example where convergent evolution has occurred in enzymes

A

chymotrypsin is a mammalian serine protease and subtilisin is a bacterial serine protease. they have very different structures but similar mechanisms

226
Q

how is enzyme activity calculated

A

see summary notes for calculation example

227
Q

what is enzyme kinetics

A

the study of how fast an enzyme catalyses its reaction and what factors affect this - it can provide information on mechanism and function

228
Q

what is the linweaver burk plot

A

1/V vs 1/[S]

- it can be used to measure Vmax and Km

229
Q

what is turnover number

A

kcat or k2
it is how fast an enzyme can work
the number of molecules of S–>P by one enzyme active site at substrate saturation per unit time

230
Q

the initial rate of an enzyme catalysed reaction increases with …….. ……………

A

substrate concentration - the curve get closer and closer to Vmax

231
Q

in an uncatalyzed reaction rate increases …………. with [S}

A

linearly

232
Q

what is the michaelis menten equation

A

V = (Vmax[S])/(Km +[S])

where E + S (K1 forward, K-1 back) ES –> (K2) E +P

233
Q

what is Vmax

A

the reaction rate when the enzyme is fully loaded with substrate

234
Q

Vmax is directly proportional to the ………… …………..

A

enzyme concentration

235
Q

what is Km

A

the substrate concentration required to give Vmax/2

236
Q

Km is dependent/independent of [E]

A

independent

237
Q

what is the michaelis menten reciprocal

A

1/v = 1/Vmax + Km/Vmax x 1/[S]

1/Vmax is where the line crosses the Y axis

238
Q

what is the equation for Kd

A

Kd = koff/kon

239
Q

what is the equation for Km

A

Km = (k-1 +k2)/k1

240
Q

when would Km = Kd

A

when the catalysis is very slow relative to the binding and dissociation

241
Q

explain how Km values are relevant to function

A

control of glycolysis in the liver and muscle cells are different
the liver is freely permeable to glucose so cytosolic [glucose] varies around 5mM according to fed/fasted states. Glucokinase Km ~ 10mM
muscle is not freely permeable to glucose soo cytosolic [glucose] is low. hexokinase Km ~0.1mM

242
Q

…….….. …………….. can bind activators or inhibitors. these enzymes often show ……….. kinetics, not hyperbolic kinetics

A

allosteric sites

sigmoid kinetics

243
Q

phosphofructokinase is allosterically activated by …………. and ………… and inhibited by …………. and ……………….

A

F 2,6 BP and AMP

ATP and citrate

244
Q

in the liver F 2,6 BP is made when blood glucose is high/low which controls glycolysis

A

high

245
Q

at saturation what does Vmax equal

A

Vmax = Kcat[Etotal]

246
Q

how is turnover number calculates

A

see notes for example calculation

247
Q

what is Kcat/Km

A

the specificity constant - how efficiently does an enzyme convert its substrate to product

248
Q

lactate dehydrogenase has very high/low malate dehydrogenase activity. why is this and how can we change it

A

low
malate has one more Ch2 group and one more -ve charge than lactate
there are 3 mutations (single amino acid changes that increase active site volume, remove a -ve charge and add a +ve charge) which together convert lactate dehydrogenase to malate dehydrogenase by redesigning the enzyme specificity

249
Q

lower Km = higher/lower affinity

A

higher

250
Q

higher Kcat = better/worse catalysis

A

better

251
Q

what happens when NAD binds to lactose dehydrogenase

A

it involves many interactions i.e. ionic interactions, H bonds, hydrophobic interactions

252
Q

why is NADP+ not a substrate of lactose dehydrogenase

A

interaction of 2’-OH explains why NADP+ isn’t a substrate. NAD enzymes cant use NADPH and vice versa

253
Q

describe competitive inhibitors

A
  • they resemble the substrate and bind reversibly (weakly/non-covalently) to the active site
  • increase Km by a factor of 1 + [I]/Ki
  • there is no change in Vmax
  • with inhibition 1/V is higher so rate is lower
254
Q

describe irreversible inhibition and give an example

A
  • inhibitors react covalently with active site group essential for catalysis
  • chymotrypsin is irreversibly inactivated by reaction with DIPF which reacts with ser195. the OH of serine reacts with DIPF causing fluoride displacement and covalent bond formation between DIPF and ser195
255
Q

why do we know that the enzyme stabilises the transition state

A

it binds to it better than the substrate or product
it lowers the transition state energy level
this is how Ea is reduced

256
Q

name 5 other ways in which a chemical reaction can be accelerated other than stabilising the transition state by reducing Ea

A
  • acid-base catalysis
  • acid catalysis
  • base catalysis
  • covalent catalysis
  • metal ion catalysis
257
Q

what is acid-base catalysis

A

speeding up the reaction by residues with charged side chains (also N/C terminal)
the acid or base is not itself consumed in the chemical reaction

258
Q

what is acid catalysis

A

donation of a proton

259
Q

what is base catalysis

A

accepting a proton

260
Q

what is covalent catalysis

A

Nu attack by unprotonated His, Lys, OH, SH, COO- to produce a covalent intermediate which is transiently covalently attached to the enzyme - provides an alternate pathway to an uncatalyzed reaction

261
Q

what is metal ion catalysis

A

metal ions stabilise the developing -ve charge on an O atom in the transition state

262
Q

describe he amide hydrolysis transition state

A

when the OH and the O are both bound to the C and the NH2 leaving groups is also still attached

263
Q

how are catalytic Abs used in amide hydrolysis

A

the antigen is a transition state analogue and the Abs against it hydrolyse the amide, increasing uncatalyzed rate dramatically. the rate is increased but not to as much as if the native transition state was used

264
Q

apoenzyme (protein portion) + cofactor (non protein portion) –> ………………..

A

haloenzyme (whole enzyme)

265
Q

what is pyridoxal phosphate

A

cofactor required for catalysis by glycogen phosphorylase, aminotransferases and other enzymes

266
Q

what are chymotrypsin, trypsin and elastase

A

they are proteases stored as zymogens in the pancreas and are activated by proteolysis on release into the small intestine
they all use the same mechanism as water hydrolysis

267
Q

what is the role of chymotrypsin

A

it hydrolyses peptide bonds after aromatic amino acids

268
Q

what is the role of trypsin

A

it hydrolyses peptide bonds after basic amino acids

269
Q

what is the role of elastase

A

it hydrolyses peptide bonds after small neutral amino acids

270
Q

what does DIPF do to chymotrypsin

A

it reacts with ser195 to inactivate chymotrypsin

271
Q

what are the key structures of chymotrypsin

A

Asp 102
His57
ser195

272
Q

what are the six steps in the chymotrypsin mechanism

A
  1. ser195 of the chymotrypsin protease attacks the carbonyl group of the substrate. His57acts as a base catalyst, removing the proton from the ser OH group to form an alkoxide ion
  2. the oxyanion (alkoxide) intermediate is stabilised by H bonding to the backbone groups in the oxyanion hole
    - covalent catalysis - enzyme covalently attached to substrate
    - TS stabilised
    - only get bonding with transition state
    - O- stabilised by H bonding
  3. C-N bond is broken. N terminal part (R1COO-) is attached to ser195 - the acyl enzyme. C terminal part (R2NH2) is still in the active site. H bonded to His57
    - acid catalysis
    - NH2 is part of the product (replaced by water)
    - acyl enzyme - substrate acyl transiently attached to serine hydroxy group of enzyme
  4. the C terminal part of the substrate leaves
  5. H2O replaces the C terminal part and attacks the acyl enzyme
  6. the second oxyanion intermediate is established by the oxyanion hole, then His57 acts as an acid catalyst to release the N terminal product
    (try learn this but use the full wiki mechanism for understanding)
273
Q

what is a catalytic triad

A

group of 3 amino acids found in the active sites of proteases, involved in catalysis. it is a common motif for generating a Nu residue for covalent catalysis (ser is the Nu)

274
Q

what does the catalytic triad do

A

it removes a proton from ser195 making it a very strong Nu. ser195 becomes covalently attached to the substrate in the acyl enzyme (covalent catalysis)

275
Q

the ………. ………… makes favourable interactions with the oxyanion hole that neither the substrate or product can make

A

transition state

276
Q

how does the substrate binding pocket determine substrate specificity

A

the specificity pocket binds the sidechain and the pockets are specific in that they will only bind certain sidechains

277
Q

describe the specificity pocket of the 3 proteases

A

chymotrypsin - made of np groups that make hydrophobic interactions - aromatic side chain can fit
trypsin - aspartate vesicle chain only accepts +ve side chains
elastase - only fits small hydrophobic sidechains

278
Q

what are statins

A

competitive inhibitors of HMGCoA reductase that lower cholesterol and are used in prevention/treatment of heart disease

279
Q

what is the normal pathway in the production of cholesterol and where do statins act

A

acetyl coA –> HMGCoA –> mevalonate –> cholesterol

statins act in the conversion to mevalonate by blocking the HMGCoA reductase

280
Q

describe nerve gases

A
  • irreversible inhibitors
281
Q

what is the effect of sarin

A

it is a nerve gas

  • it inactivates acetylcholine esterase at neuromuscular junctions and looks similar to DIPF which inactivates chymotrypsin
  • acetylcholine esterase breaks down neurotransmitters allowing muscles to relax but sarin inhibits this causing constant contraction making you unable to breath
  • sarin reacts with one ser residue
282
Q

what are the 2 types of serine protease

A

chymotrypsin of subtilisin like

283
Q

how do artificial arms work

A

they feed signals to the brain that allow movement

284
Q

how can frog muscles be active outside the body

A

when they are brushed with different solutions this results in action potentials being generated

285
Q

what happens when a plant is wounded

A

an electric signal is transmitted through the entire plant

286
Q

separated charges have ………… ……..

A

potential energy

287
Q

ions on opposite sides of the membrane with opposite charges create ……….. …………

A

membrane potential

288
Q

describe the resting membrane potential

A

it is negative inside cells

289
Q

how can resting potential be measured

A

using a voltmeter - one electrode in the cell and one in the extracellular fluid

290
Q

what is diffusion

A

the net movement of molecules from high concentration to low concentration due to thermal motion

291
Q

…….….. particles reach equilibrium before we have equal concentration

A

charged

292
Q

if there is a concentration gradient for X+ and the membrane is selectively permeable for X+ there will always be an ………… ………….. across the membrane

A

electrical potential

293
Q

describe the main concentration gradients in animal cells and plant cells and what is the basis for these

A

animal - less Na inside than out, more k, less Cl, more organic components
plant - more Na inside than out, more K, more Cl, more organic components
they are all based on NaK and ATP proton pumps

294
Q

ATP pumps protons in/out of the cell

A

out

295
Q

what are the 3 types of secondary transport

A

antiport, symport, cotransport

296
Q

if the membrane is 100% selective for K+ then the membrane potential = ………… ……………..

A

equilibrium potential

297
Q

concentration gradient does/doesn’t matter if the membrane isn’t permeable

A

doesn’t matter - voltage depends only on those things that are permeable

298
Q

what is the Nernst equation and what is it used for

A

it describes the equilibrium potential for an ion at room temperature
Ex = Z(-60mV)log10([Xin]/[Xout])
see notes for example

299
Q

what is the PM more permeable to Na or K

A

K

300
Q

what si the membrane potential and why

A

resting potential for a typical neuron is -60mV this is closer to the equilibrium potential for K (-90) than Na (+60) because the membrane is more permeable to K so it has more of an effect on the membrane potential

301
Q

membrane potential is determines by the …… ……….. and the ………….. of each ion

A

equilibrium potential

permeability

302
Q

membrane potential can be represented as a circuit - describe how this is shown

A

permeability = conductance (1/resistance)
equilibrium potential = battery
voltage across the circuit measured by batteries in parallel
conductance changes - changes membrane potential
ion channels are tuneable conductors

303
Q

describe the action potential generation

A

generated by transient changes in Na and K permeability due to opening and closing of channels

  1. resting - more K open than Na channels (-ve)
  2. depolarisation - opening of Na channels (-ve –> +ve)
  3. repolarisation - closure of Na and opening of K channels (back to -ve)
  4. hyperpolarisation - K channels remain open after resting potential reached
304
Q

what direction does Na move in through the Na channels

A

into the cell

305
Q

what direction does K move in through the K channels

A

K moves out

306
Q

Na/K channels are stronger in defining the final voltage

A

Na

307
Q

what is patch clamp and describe the method

A

it is a method used to study ionic current sin individual isolated living cells, tissue sections or patches of a cell membrane (even individual channels)
- fill pipette with solution connected to electrode with amplifier. Put pipette on top of cell and suck - seal created between electrode and membrane - take measurements and see channels opening and closing from the readings

308
Q

what are the 3 different modes of patch clamp

A

cell attached, whole cell or excised patch

309
Q

what is membrane potential

A

the difference between the electrical potential of interior and exterior of the cell - determined by the equilibrium potential and permeability of each ion

310
Q

what creates a concentration gradient

A

primary pumps and secondary transport

311
Q

alpha helices allow proteins to arrange amino acid sidechains so that they …………. ……… ………. ………… but these structures on their own are not enough to constitute a …………….. ………………… because soluble proteins also have alpha helices

A

compensate each others charge

transmembrane protein

312
Q

what does the hydropathy index indicate

A

the hydrophobicity of an amino acid (kJmol-1). it gives the energy for the transfer of an amino acid from a hydrophobic to a hydrophilic environment
-ve - release energy - hydrophilic
+ve - invest energy - hydrophobic
0 - amphiphilic

313
Q

describe the Na channel topology

A

24 Tm domains each consisting of a large number of hydrophobic amino acids linked by hydrophilic linker domains. there are 6 domains per repeat. between 5 and 6 the protein dips into the membrane

314
Q

what does the gradient of a plot showing current vs voltage tell us

A 
steep = high conductance 
flatter = low conductance
315
Q

what does excised patch allow for

A

individual channel current measurement, control of ion concentrations on both sides of the membrane, and control of membrane potential

316
Q

what is reversal potential

A

voltage where current = 0. we compare it with the equilibrium potential to infer selectivity of an ion for a particular channel

317
Q

what is open probability

A

frequency of opening

318
Q

what changes the open probability

A

gating - by voltage (inward rectifying, outward rectifying) or ligands

319
Q

describe the shaker K channel

A

only one 6 domain unit

320
Q

describe the prokaryotic transmembrane channel

A

has domains S5-6 only so only has pore (between 5-6 the protein dips into the membrane - selectivity filter

321
Q

describe the K channel pore and how ions pass through

A

3 Armstrong’s in diameter which is wide enough for K and Na but is still selective for K. 4 units of protein determine the size of the pore. the narrowest part is determined by the partial transmembrane domains. the ions need to strip off their hydration shell t pass through the pore

322
Q

which ion is smaller Na or K and how does size affect the hydration shell

A

Na is smaller and binds to water more tightly due to the charge being more concentrated in a smaller ion

323
Q

what is the TVGYG motif

A

it is a well conserved selectivity filter favourable for K

324
Q

why is the K channel selective for K and not Na

A

K gains more energy in the domain than is used to remove the hydration shell (for Na its the opposite) so the pore is favourable for K and not Na

325
Q

how many binding sites does K channel have and how is this useful

A

4 binding sites - ions push one another up one site due to the electrostatic repulsion. there are K in the binding sites a any one time. high rate of transport

326
Q

the K channel is voltage/ligand gated - what triggers it to open

A

voltage - it opens due to depolarisation. when voltage changes the domains change which leads to opening or closing of the pore

327
Q

why do we need to look at the mammalian K channel to study voltage gating

A

without all 6 domains we don’t see voltage gating (prokaryotic channel only has the pore domains)

328
Q

the S4 domain has a lot of …….. amino acids and can sense voltage change because of this

A

charged

329
Q

how is the K channel inactivated

A

the N terminal is critical. when voltage stays the same the current still disappears. the last 20 amino acids are positively charged and attached to a flexible segment of the polypeptide chain. when the channel opens the ball (20aa) is attracted to the pore and occludes it. the channel only conducts for a short period of time

330
Q

describe the acetylcholine receptor

A

it is a ligand gated ion channel gated by acetylcholine. it is a non selective cation channel. it is a pentameric channel with transmembrane domains. 5 units come together to form the pore

331
Q

describe acetylcholine receptor channel opening and what effect does this have on the cell

A

channel opening results in a small depolarisation of the membrane potential which triggers an action potential. as action potential arrives at the neuron end, this leads to acetylcholine release from the synaptic vesicles at the synaptic cleft. receptors in the next neuron perceive the signal, opening the channel, generating an action potential in the neuron

332
Q

describe the difference in structure between the open and closed acetylcholine receptor

A

closed - 2 rings don’t fit well into each other

open - twisting mechanism caused by acetylcholine - achieved by rotating M12 TM helix by 15 degrees

333
Q

give an example of a P-type ATPase

A

Ca ATPase (SERCA) - pumps Ca from the cytoplasm into the sarcoplasm reticulum of muscle cells

334
Q

muscle contraction is triggered by low/high Ca concentration in the muscle cytoplasm

A

high

335
Q

how do we get muscles to relax after contraction

A

Ca is pumped from the cytoplasm into the SR which requires ATP

336
Q

describe the process of muscle contraction initiation in terms of the Ca pump

A

action potential arrives and the Ca channel opens due to being voltage gated. Ca rushes into the cytoplasm and this results in muscle contraction. this is very quick because initial cytoplasmic Ca is very low
ATP –> ADP at the N domain and phosphate is transferred to the P domain (Asp351). ATP binding and phosphorylation rearranges the A, P, N domains and subsequently domains so that 2Ca are released into the SR. dephosphorylation of the P domain converts protein to its original state

337
Q

High/low Ca affinity (take from cytoplasm) –> (conf change) High/low Ca affinity (go into SR)

A

high

low

338
Q

name and describe a method used for structure-function analysis

A

mutation and electrophysiological experiments e.g. heterologous expression of membrane proteins in Xenopus oocytes

  1. clone channel in E.coli
  2. carry out in vitro transcription
  3. inject RNA into oocytes and measure ion currents - the protein gets put into the membrane by the oocyte
339
Q

the citric acid cycle harvest electrons through …….. which are then fed into the ETC to set up a ……….. ……….. used by …………… …………. to make ATP

A

NADH
proton gradient
ATP synthase

340
Q

where do the sugars in our food come from

A

they ultimately come from that produced by plant photosynthesis

341
Q

how is excess glucose stored

A

glycogen
starch
fat
proteins etc

342
Q

what is the main plant store of excess glucose

A

starch - can be converted to ATP when required

343
Q

how do bacteria and lower organisms adjust their work/energy supply balance

A

they detect nutrient availability in the environment. they move towards nutrient and can adjust reproduction and metabolism

344
Q

how do plants adjust their work/energy supply balance

A

they adjust metabolism/growth in response to nutrient availability

345
Q

how do higher organisms adjust their work/energy supply balance

A

they detect internal nutrient/energy status and environment status

346
Q

what is another name for transporter receptors

A

transceptors

347
Q

describe chemoreceptors of prokaryotes

A

they are usually histidine kinase systems and detect and respond to ligands and adjust accordingly

348
Q

what are 2 roles of the plant vacuole

A

store of nutrients and the site of autophagy

349
Q

what is autophagy

A

breakdown to recover building blocks

350
Q

what organelle of animals acts in a similar way to the vacuole of plants

A

lysosomes

351
Q

as well as the current and surrounding levels, higher animals can sense ………… levels of nutrients and adjust to changes

A

storage

352
Q

does a high energy status promote anabolic or catabolic pathways

A

anabolic

353
Q

what does AMPK detect

A

the AMP : ATP

354
Q

how do we respond to high AMP

A

high AMP –> AMPK activated –> stimulate catabolism –> energy homeostasis

355
Q

how do we respond to low AMP

A

low AMP –> AMPK not activated –> stimulate anabolism –> energy homeostasis

356
Q

what happens to ATP levels when the first ETC protein complex is blocked with berberine and how does this compare to what is expected and explain the situation

A

we expect reduced ATP and ADP build up. the ATP decrease is actually minor and the ADP increase is lower than expected. we see a large AMP increase. adenylate kinase maintains ATP in the short term by transforming ADP to ATP and AMP

357
Q

what is the chemical equation for transforming ADP to AMP and ATP

A

2ADP –> ATP + AMP when ADP:ATP high
ATP + AMP –> 2ADP when ADP:ATP low
catalysed by adenylate kinase

358
Q

AMP detection rather than ATP allows very early detection of ATP production proteins, why is this

A

AMP is increasing before ATP levels go down. the early change is AMP increase and the late change is that ATP goes down

359
Q

what happens when AMP:ATP is high

A

AMPK is activated by being phosphorylated by an upstream kinase, and upregulates the energy supply and downregulates energy expenditure

360
Q

describe AMPK structure and the function of each of the subunits

A

it is a heterotrimer
- alpha subunit - catalytic - contains the kinase domain and is the site where AMPK is phosphorylated (conformation change moves the autoinhibitory domain away from the catalytic domain to activate the enzyme)
- beta subunit - linker - detects other types of stimuli
- gamma subunit - regulatory - binds ATP, AMP, ADP
the subunits are each encoded by their own gene

361
Q

describe the triple mode of AMPK activation

A
  1. AMP binds, promoting AMPK thr172 phosphorylation by LKB1
  2. binding of AMP inhibits thr172 dephosphorylation
  3. AMP binding causes allosteric activation
    all 3 effects are antagonised by ATP
362
Q

why isn’t AMPK regulation described as on/off

A

because expression is controlled/regulated according to the AMP/ATP ratio

363
Q

list some AMPK outputs

A

signalling pathways regulate processes in glucose metabolism, lipid metabolism, protein synthesis, anti-inflammatory, anti-ageing, redox regulation

364
Q

list some inputs for AMPK

A

targeted by signalling pathways e.g. calorific restriction, overnutrition, obesity/inflammation, and exercise/contraction. some involve AMP/ATP pathways but other don’t

365
Q

what is SnRK1

A

it is found in plants and is a kinase very similar to AMPK. it regulates energy metabolism in plants. it is partially regulated by AMP and directly targeted by environmental stress. it helps balance how much energy they put into growing and defence

366
Q

insulin detects …… ………

A

blood sugar

367
Q

what happens during long term starvation

A
  1. decrease in blood glucose and increase in glucagon and glycogenolysis. once glycogen is depleted we see gluconeogenesis. glucose prioritised to the brain and ketone bodies to other tissues. BMR increases for a period of hyperactivity then decreases again
  2. ketone bodies used by the brain. less energy expenditure, body temp, heart rate, bp and respiration, brain activity, protein synthesis, immune activity. muscle weakness. GIT organ atrophy
  3. loss of 40-50% body weight. proteins of organs, muscle, cell membrane, blood are used for energy. see dehydration, oedema, cardiac arrhythmia. paralysis –> death
368
Q

in plants starch breakdown occurs when and how is it adjusted

A

at night
it is adjusted to the amount of starch made during the day. they use all the starch before the next day to optimise growth. the starch degradation is adjusted to anticipate night length - its adjusted to the 24h cycle

369
Q

what happens if plants use up their starch at night too early

A

if used too early they will starve and there will be a delay in starch production the next day

370
Q

many plants mutants in the use of starch at night also have defects in the ………… ……….

A

circadian clock

371
Q

what chemically promotes breakdown of starch in plants

A

phosphorylation of the starch granule

GMC (4 decks)

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