Biostructures, Synthesis, energetics Flashcards
What is the tendency of entropy?
To always increase.
What is the “central dogma of energy conversion”?
Release -> Transfer -> Trapping
What is delta P?
The proton motive force.
What are reductants?
Molecules broken down via metabolism to release electrons -> transferred via coupled reaction with protons pumps (redox) -> force to synthesise ATP
What causes a motive force?
Displacing reactions from equilibrium.
What is the function of Transhydrogenase?
Synthesise reductants
What photosystems are used by phototrophs?
Photosystems 1 + 2
What are the 3 pieces of evidence for Chemiosmotic Theory:
-Jagendorf Acid Bath
-DNP addition
-Isolated Bacteriorhodopsin with Beef Heart ATP Synthase.
Jagendorf Bath Experiment.
thylakoid incubated a pH 4 in dark (using weak permeable acid e.g. succinate) -> Transferred to high pH medium -> forms deltaP -> Allows for ATP synthesis when ADP and Pi are added.
DNP as evidence for chemisomosis:
DNP acts as an uncoupler, it facilitates the diffusion of protons across the membrane -> prevent formation of pmf -> abolishing ATP synthesis.
Bacteriorhodopsin reconstitution as evidence for chemiosmosis:
The pump is isolated and reconstituted onto a lipid vessicle with ATP synthase from beef heart -> in presence of ADP and Pi -> ATP synthesis can occur.
Where do dark reactions occur?
In the stroma.
What is the generic role of chlorophyll?
Light Harvesting
Similarity between chlorophyll and Haem?
-Both have a tetrapyrrole ring (similar to benzene ring having a conjugate pi electron system)
- Both have a prosthetic group (Mg2+ and Fe2+)
What structure in chlorophyll is responsible for light absorption?
The tetrapyrrole ring tuned by Mg2+
Where do the light reactions take place?
In the Thylakoid Membrane (ATP synthase, PSII, PSI, Cytochrome b6f)
What occurs in a chlorophyll pigment upon absorption of light?
An electron in the tetrapyrrole ring is excited to a higher energy level.
What is the Q cycle process?
The two different routes of electrons from Plastoquinol in the cytochrome b6f complex.
Where is plastoquinone and ubiquinone
Plastoquinone is in the thylakoid, Ubiquinone is in the mitochondria.
What does endergonic mean?
“uphill” reaction requiring an input of energy.
What is the special pair of chlorophylls called in PSII?
P680
What is the special pair of chlorophylls called in PSI?
P700
What does PSII oxidise and reduce?
Oxidises Water
Reduces Plastoquinone into plastoquinol
What does PSI oxidise and reduce?
Oxidises plastocyanin and reduces ferrodoxin -> which donates electrons to NAD+ -> NADPH
What are the products of water oxidisation?
2H2O -> O2 + 4H+ + 4e-
What is the effect of exciting the special chlorophyll pair?
Gives it a more negative redox potential, allowing it to donate its electron to downstream components in the electron transport chain.
What is the order of components in PSII?
P680 -> Plastoquinone (reduction) -> Cytochrome b6f -> Photosynthetic complex -> P700 (PSI) -> ferrodoxin -> NADP+
What is the structure of a photosystem?
An antenna complex of hundreds of accessory chlorophyll pigments that transfer light energy to the reaction centre to a special pair of redox active chlorophyll.
What is the purpose of oxidising in PSI and PSII?
To restore the electron lost by the photosystem.
What is the purpose of the antenna complex within photosystems?
To concentrate light in the reaction centre to maximise electron excitation within turnover capacity. (despite shade)
How does the R-group on the tetrapyrrole ring differ between Chlorophyll a and chlorophyll b?
A has a methyl group and B has a CHO (aldehyde) group
What is the effect of the differing R-groups on chlorophyll a and chlorophyll b?
They affect the electron environment, affecting the absorbance spectrum, therefore the combination of both allows for the absorption of a wider range of wavelengths.
What is the antenna structure of PSII?
PSII forms a dimeric super structure with Light Harvesting Complex II (LHCII) -> multiple antenna proteins provide large spatial cross-section for light absorption.
What is the antenna structure of PSI?
PSI forms a monomeric supercomplex with Light Harvesting Complex I -> multiple antenna proteins provide large spatial cross-section for light absorption.
What is meant by photosystem antenna structures being modular?
The LHC can grow more/less depending on the conditions. (more under low light, less under high light).
What do shapes on light spectra correspond to?
The jumps electrons make between orbitals (energy levels)
What is the oxygenic evolving complex?q
Site of water oxidation attached to reaction centre of PSII
What causes plants to fluoresce?
After time excited electrons will re-enter the ground centre, emitting light.
What photons are absorbed by molecules?
Photons with energy equal to that of the energy gap between electronic states.
(Blue S0 -> S2 and Red S0 -> S1)
How quickly does light absorption occur?
On a femtoseconds (10^-15s)
What is FRET?
Forster Resonance Energy Tranfer
What is the process of FRET?
When two chlorophylls are in close proximity, excited electrons in overlapping energy levels -> allow for electron in S1 to donate energy to a neighbouring chlorophyll molecule, exciting its electron to S1.
In what forms is energy lost as in chlorophyll after electron excitation?
Lost as heat (S2 -> S1) or as vibration or fluorescence emission (S1 -> S0) (stoke shift as light emission is more red than light absorbed)
Over what distances is FRET efficient?
Short distances of 7nm<
What is resonance?
Overlapping energy levels causing a transfer of energy.
What is the purpose of FRET?
Provides a downhill pathway for electron excitation to be directed to the Reaction Centre whilst minimising energy loss.
What is responsible for the directionality of the attenae complex?
The environment surrounding the chlorophyll and their bound r-groups affects the energy of their S1 excitation levels, pigments with lower energy levels are positioned closer to the RC.
Light Harvesting Complex 2: Structure
-Hydrophobic and Uniform.
-4x carotenoids
-6x Chlorophyll b
-8x chlorophyll a
What is the redox potential required for the oxidation of water?
Atleast +820mV
What is the reaction that occurs at Photosystem II?
2H2O + 2PQ + 4H+(stroma) -> O2 + 2PQH2 + 4H+(lumen)
What is the primary electron donor in the reaction at PS II?
The special pair chlorophyll P680
What is efficiency of the PSII reaction?
40%
What are the key co-factors that take part in the electron transfer reactions from water to plastoquinone? (PSII)
Chlorophyll aa and chlorophyll ab -> pheophytin (a + b), plastoquinone (Qa + Qb), tyrosine 161, manganese cluster.
How does the electron travel to reduce plastoquinone in PSII?
A chlorophyll a undergoes charge separation upon the induction of light energy at the reaction centre -> the electron moves towards stromal side across the co-factors onto plastoquinone Qb. The electron hole on the chlorophyll a is filled by water bound to the manganese cluster. -> process repeats -> and once two electrons have attached to plastoquinone Qb -> plastoquinol will dissociate.
What is the purpose of the manganese cluster in PSII?
To catalyse the oxidation of water (hydrolysis)
What is the role of tyrosine 161?
To transfer an electron from the manganese cluster to the RC to fill the electron hole.
How does the manganese cluster oxidate water?
It accumulates a +4 charge as it donates electrons to the P680* ->
What groups of plastoquinone are reduced?
Two carbonyl groups either side of a hex-2,5-diene
What is the function of plastoquinone’s large tail?
to aid its lipid solubility.
How many turnovers of the PSII are required for one full reaction?
2 turnovers (4 photons)
Other than the reduction of Plastoquinone into Plastoquinol, what is the additional function of PSII?
The pumping of H+ from the stroma into the lumen.
What is the shape/arrangement of cofactors in PSII?
A horse shoe arrangement.
What is the name of the combination of a photosystem and light harvesting complex?
a supercomplex.
What structural feature of PSI prevents the escape of electrons from the RC?
Neutral zone surrounding the RC.
What is the efficiency of the PSI reaction?
44%
What is the overall reaction of PSI?
Pc(red) + Fd(ox) -> Pc(ox) + Fd(red)
What is the electron donor in PSI?
Plastocyanin.
Structure of Plastocyanin:
2x Histeine, Cysteine, and methionine residues coordinate the active site
What is the redox potential of the P700* centre in PSI?
-1320 mV -> sufficient to reduce 2Fe-2S cluster of ferredoxin (-420 mV)
What are components of PSI?
The special pair of chlorophylls, Phylloquinone, and iron-sulphur clusters.
What is the role of reduced ferredoxin?
Acts as a powerful reductant that can reduce NADP+ into NADH and NO3 into NH4.
What is Marcus Theory:
An explanation of the rates of ET reactions where participants don’t undergo large changes -> smaller gradual changes less thermodynamically feasible occur more quickly than one very feasible larger step.
What is the redox potential of the P700 centre in PSI?
+480mv -> this is insufficient to oxidise water.
What is reorganisation energy?
When an electron is transferred the molecules around the donor/acceptor have to move to accommodate the change of charge -> energy is used to do this.
When is the rate of electron transfer optimal?
When reorganisation energy = delta G
How is energy loss by heat minimised in reaction centres?
The e- and +ve hole are quickly separated physically and energetically to prevent their recombination and ensuing heat loss.
Water oxidation cycle:
2H2O -> HO + H2O -> HO + H2O -> HO + HO -> HO + HO -> H2O + H2O
What occurs at each step of the water oxidation cycle? (to the manganese cluster)
The manganese cluster loses an electron each step, until it’s regenerated via the oxidation of Water.
What is inputted to the water oxidation cycle to regenerate it?
2 H2O molecules.
How many electrons are released in one cycle of the water oxidation cycle?
4 electrons.
What are components of Cytochrome b6f?
(2x plastaquinone, 1x carotenoid, 1x chlorophyll, 4 haem groups, 1 2Fe2S cluster } Per monomer in complex dimer
What is cytochrome b6f similar to?
Cytochrome in mitochondria.
What is the purpose of the carotenoid and chlorophyll portion of cytochrome b6f?
No clear purpose as cytochrome doesn’t use light harvesting.
What are the redox active cofactors in Cytochrome b6f?
Haem groups and 2Fe”S clusters.
What is the function of redox active cofactors in an electron transfer chain?
To carry the electron across through complexes.
What are the two types of haem groups in cytochrome b6f?
c-types (covalently linked to proteins via cysteine side chains)
b-types(linked via coordination bonds usually from histidine lone pair on nitrogen ligates to central Fe iron of haem group)
What is Bifurcated electron transfer?
The electrons are transferred but take two different paths, occurs in cytochrome b6f.
What type of electron carrier is plastoquinol?
A 2 electron 2 proton carrier.
At cytochrome b6f what and where does plastoquinol reduce?
Plastoquinol reduces the 2Fe2S cluster and the Haem Bp
What is the destination of the electron transferred to the 2FE2S cluster in cytochrome b6f?
Haem f (c-type)and then reduces Plastocyanin (ox) into Plastocyanin (red)
What type of electron carrier is plastocyanin?
A 1 electron carrier.
What is the high potential chain of cytochrome b6f?
The reduction of plastocyanin.
What is the low potential chain of cytochrome b6f?
The reduction of a stromal plastoquinone molecule into platoquinol (occurs after two runs)
What molecule in plastocyanin allows for it to accept electrons?
Cu centre.
What is the destination of the electron transferred to the Haem Bp in cytochrome b6f?
The electron is then transferred to Haem Bn and then Haem C -> Will then transfer to stromal plastoquinone after two runs.
What is the function of recycling one of the electrons from Plastoquinol to form plastoquinol at cytochrome b6f?
It doubles the number of protons transferred from the stroma to the lumen per PQH2 oxidised -> boosting its contribution to the pmf.
What the Ferredoxin-NADP+ reductase’s 2 domains?
-FAD binding domain
-NADP+ binding domain
What is an FAD domain?
A flavin adenine dinucleotide.
What is the name of the 3rd protein complex in the electron transport chain in the thylakoid membrane?
Ferredoxin-NADP+ reductase (FNR)
What are the steps of NADP reduction at the FNR?
- Binding of NADP+ and Fd to binding domains
- Fd donates electron to FAD and disociates x2
-The two electrons reduce NADP+ -> recruits a proton to form NADPH -> disociates.
What is the proton/ATP ratio in the chloroplast?
4.67
How many ATPs are formed per NADPH in photosynthesis?
1.28, however this needs to be 1.5 for the efficient functioning of photosynthesis.
How is the redox imbalance of photosynthesis (ATP/NADPH) corrected?
Redox balance -> a second type of electron transport takes place (cyclic electron transport).
What are the two complexes that participate in cyclic electron transport?
PGR5 and the photosynthetic complex 1.
What is the function of Photosynthetic complex 1 in redox balance?
allows for electrons from ferredoxin to re-enter the electron transfer chain; this is coupled with the pumping of protons into the lumen.
How do electrons on ferredoxin re-enter the electron transport chain?
The electrons are used to reduced plastoquinone into plastoquinol.
What type of enzyme does photosynthetic complex I operate as?
A ferredoxin-plastoquinone reductase.
How does photosynthetic complex I transfer electrons from ferredoxin to Plastoquinone?
Photosynthetic Complex I has 4 4FE4S clusters which are used to shuttle electron from the ferredoxin to plastoquinone.
What are the two components that form ATP synthase?
Water soluble F1 and membrane integral F0.
What is delta psi?
The difference in membrane potential across a membrane.
What equation relates delta psi, delta p, and delta pH?
Delta p = Deta psi - 60 x delta pH.
How is the membrane potential maintained despite proton pumping?
The pumping of counter ions to partition/change. (This will negatively effect the proton motive force)
What is the function of the peripheral stalk in ATP synthase?
Acts as a dynamic scaffold to hold the F1 head in place around the central rotating shaft.
purpose of the 8-15c subunits in F0 of ATP synthase?
Act as the proton driven rotor embedded in the membrane
What is the gamma subunit of ATP synthase?
The central shaft that transmits toque from F0 to F1, changing the conformation of the beta subunits.
What are the 3 conformations of Beta subunits on F1 head of ATP synthase?
Open, Tight, and Loose.
What subunits form the F1 head of atp synthase?
3 alpha and 3 beta subunits.
What is the difference between a and b subunits on ATP synthase?
They are structurally similar however only B units are catalytically active.
What is the function of alpha subunits in F1 ATP synthase?
Structural role in the F1 head.
What forms the peripheral stalk of ATP synthase?
B2 and sigma subunits.
What is the function of alpha subunits in F0:
Act as a barrier.
What is the function of c subunits in the F0 of ATP synthesis:
Acts as a water wheel, each subunits has a glutamate which binds to protons and translocates them across the membrane.
What is the function of arginine residues in the alpha subunits in F0 of ATP synthase?
Has an amine froup that can bind to protons, when in proximity to a glutamate it stabilises it, allowing for it to unprotonate the glutamate, allowing for the release of protons in the low high pH region.
What process is coupled in the F0 region of the ATP synthase?
the resolving concentration gradient with the generation of torque.
How many ATP molecules are synthesised by full rotation of the c-ring in ATP synthase?
3 ATP molecules.
What does the rotation of the gamma shaft cause in the F1 head?
It causes changes in conformation of the B-subunits.
ATP Synthase: Open State
ATP is released and ADP and Pi bind to the b subunit
ATP synthase: Tight State
ADP + PI is converted to ATP
ATP Synthase: Loose State
ADP + Pi interact with eachother
Why do animals have smaller c rings than plants?
Animals have a regulated energy input, whereas plants energy input varies massively, therefore plants have larger c-rings to prioritise efficiency over speed.
How can the work of ATP synthase be reversed?
Supply the enzyme with a large conc. of ATP -> dependent on balance between deltaG ATP and delta P.
What are the 3 parts of the calvin cycle?
Carboxylation, reduction, regeneration.
What is the net output of the calvin cycle?
1 x GAP per 3 x CO2.
What is the main output of the Calvin cycle?
Glyceraldehyde-3-phosphate
What is the function of GAPs?
Used as the starting point of multiple metabolic pathways in plants
What residue is at rubisco’s active site?
lysine
How is rubisco regenerated?
Lysine reacts with another non-substrate molecule of C2 to form a carbamate anion which can then bind to Mg2+ ->
What is the function of the Mg2+ group in rubisco?
Activates ribulose -1,5-bisphosphate so it can react with CO2.
Thioredoxin: function
Regulates the activity of several calvin cycle enzymes, ensuring the activity of the light and dark reactions is closely regulated.
How many regulatory subunits are within rubisco?
8 subunits.
What is the product of carboxylation?(Calvin cycle)
2 3-phosphoglycerates per ribulose.
What is reacted in carboxylation?
Ribulose with CO2 and H2O.
What is stage of Calvin cycle involves rubisco?
Carboxylation
What occurs in the 1st reaction of the reduction stage of the Calvin Cycle:
3-phosphoglycerate is phosphorylated by phosphoglycerate kinase -> form 1,3-bisphosphoglycerate.
Where are the ATP molecules in the reduction stage of the Calvin cycle sourced from?
The light dependent reactions.
What occurs in the 2nd reaction of the reduction stage of the Calvin Cycle:
NADPH reduces 1,3-bisphophoglycerate to glyceraldehyde 3-phosphate (GAP)
What enzyme mediates the reduction stage of the Calvin Cycle:
glyceraldehyde-3-phosphate dehydrogenase.
What occurs in regeneration of the Calvin cycle?
-3 molecules of 5C sugar ribulose 5-phosphate formed
-Ribulose 5-phosphate is phosphorylated by phosphoribulose kinase to regenerate 1,5-bisphosphate.
Why are enzyme active sites are in hydrophobic pockets?
To allow for the force/power of the charged residues to have an effect on the substrate. -
Threshold enzymes:
Enzymes that bring key elements to a reaction.
Example of threshold enzyme:
Pyruvate carboxylase.
Primary metabolism:
-Essential compounds
-Basic housekeeping functions present in all cells
-Synthesised all the time (constitutive)
Secondary Metabolism:
-Specialised functions
-Present in differentiated cells
-Inducible
-E.g. antibiotics
X-ray crystallography:
-Equally spaced protein isomers
-laser -> scatters through crystal
-shows amplitude but not phase
-Uses Fournier Equation
What is phase?
Phase is the spatial information of a protein
What is the fournier transformation?
An equation that can be used to deduce structure from amplification.
What is Catabolism?
Breakdown of biomolecules. -> release energy
What is anabolism?:
Build up of new biomolecules. -> use up energy
What is the effect of phosphorylation on a molecule?
Addition of Pi group -> molecule becomes a higher energy form.
Why should glycolysis and gluconeogenesis occur at the same place?
It would be an energy sink/waste, because glycolysis doesn’t produce enough ATP for gluconeogenesis.
What is one benefit of energy lost by glycolysis?
Energy released as heat.
What occurs during resting metabolism?
A mixture of glycolysis and gluconeogenesis -> occurs despite energy wastage to conserve atom economy.
Why do reactions generating CO2 tend to be energetically favourable?
Co2 is very stable. CO2 readily escapes the site of reaction, Loss of CO2 from reaction is irreversible.
How do humans fix carbon and nitrogen?
From food/consumption.
CO2 can be fixed by what reaction?
Pyruvate with ATP and oxaloacetate.
What are group carriers?
Groups linking metabolic intermediates into larger molecules to prevent the loss of intermediates and increase energy efficieny.
What can be used to observe the metabolic activity of organisms?
Metabolic flux analysis.
COO carrier:
Biotin
C5 carrier:
Isopentenyl pyrophosphate
C1 (methyl) carrier:
S-adenosyl methionine
NH2 carrier:
Glutamine
C1 (CH, Ch2) carrier:
Folic Acid
C2 carrier:
Coenzyme A
[ATP] in cell?
10mM
What are the functions of vitamins?
B vitamins -> carriers
A - Vision
C + E - Antioxidant
D - Bones
K - Blood coagulant.
Why is biotin a good carrier of CO2?
It’s nitrogen 1 is very reactive and can react with the CO2 easily. Furthermore it can be covalently linked to proteins and-so can be used to transport CO2 directly to enzymes or proteins,
What enzyme in Krebs is biotin essential for?
Pyruvate carboxylase in link reaction, biotin is essential as a CO2 carrier.
How to activate Biotin?
Biotin is reacted with a carboxyphosphate to form biotin-COO-
Why is serine an important amino acid?
It’s a key precursor amino acid in the synthesis of many other, e.g. glycine.
Purpose of Folate:
Converts dUMP inton dTMP
What is homocysteine?
Similar to cysteine but has an extra methyl group.
Epigenetics:
All cells have the same DNA however portions are silenced to differentiate function.
What is SAM?
S-adenosylmethionine.
What is a key role of SAM?
The DNA methylation of cytosine and Histones (lysines and arginines)
What groups does Acetyl Coenzyme A have?
An adenosine and a phosphate group, contains pathothenic acid, and a thioester.
Purpose of the thioester bond in Acetyl Coenzyme A?
Makes it easier to transfer the acetyl group.
What is Patothenate (B5)?
A vitamin that carries C2 units in the link reaction of glycolysis.
What vitamin is found in retinol?
Vitamin A.
C5 unit assembly:
Steroid carbon skeleton -> 4 C5 chains -> form 20 carbon chain that folds into skeleton -> form steroids.
C5 units:
3x acetyl groups are joined together and one carbon is lost as CO2.
What are C5 units often used to synthesise?
Steroids
What is released when glutamine is hydrolysed?
NH2 and glutamate.
What are the Key roles of the TCA (Krebs) cycle?
Feeds into the ETC as source of energy but also makes key intermediates for biosynthesis.
Why is fat not used as a source of energy usually?
It’s an awkward and not straightforward pathway.
What kind of process is glycolysis?
An aerobic process.
How is glucose converted into fat?
Via the addition of acetly CoA.
Why is fat a better long-term store of energy?
Has higher energy content per gram and is not soluble in water and-so therefore not easily hydrolysed.
What are the reactants in stage 1 of glycolysis?
Glucose and ATP
Where do red blood cells (erythrocytes) get their energy?
Glycolysis.
What are the products of stage 1 of glycolysis?
Two C3 units ( Glyceraldehyde 3-phosphate)
What are the intermediates in Glycolysis stage 1?
Glucose-6-phosphate (hexokinase) -> Fructose-6-phosphate (phosphoglucose isomerase) -> Fructose-1,6-bisphosphate
(phosphofructokinase) -> Glyceraldehyde-3-phosphate (Aldolase)
What do most muscles do to provide energy when not active?
Use glycolysis.
Stage 2 of glycolysis?
Pyruvate is converted to lactate (Oxidises NADH) -> liver turns lactate back into glucose.
What is a key feature of the organisation of glycolysis enzymes?
They are associated in one large complex, allowing for substrate channelling to directly transfer intermediates between enzymes.
What is an important use of glycolysis intermediates?
Used as the start of many anabolic pathways in the synthesis of amino acids.
Why is the conversion of Pyruvate into Acetyl CoA irrversible in the link reaction?
The loss of CO2 is irreversible and therefore this provides the reaction with directionality.
What is the function of Acetyl CoA?
It’s fed into the Krebs cycle (for energy production or anabolic intermediates) or converted into fatty acids.
What is the species reduced when Pyruvate is converted to lactate?
A ketone group.
Where does gluconeogenesis mainly occur?
In the liver
Are glycogen stores limited?
Yes, can run down after 90minutes of moderate activity.
What is the start point of gluconeogenesis?
Pyruvate/lactate.
What are the two sources of Pyruvate?
The transamination of alanine with alpha-ketoglutarate and glycolysis
Glutamate is a key carrier for what?
A key carrier carrier for amino groups, having an amine group that can be traded for a ketone.
In which organisms is pyruvate converted into acetaldehyde?
Yeast.
Function of the Pentose Phosphate Pathway:
Pathway that occurs in parallel to glycolysis, however has many unique intermediates that are used for biosynthesis.
What is the key functional difference between NADH and NADPH?
NADPH is a much stronger reducing agent.
Why is the used of NADH favoured in metabolism?
NADH releases less energy than NADPH, however less energy is required -> therefore less wastage.
What is an example of a key biomolecule synthesised using PPP intermediates?
Ribose sugars.
Steps of the pentose phosphate pathway?
Pentose-6-phosphate forms ribulose (loses CO2 and reduces 2NADP+ into 2NADPH) -> converted into 2 5C sugars -> 3C and 7C -> 6C and 4C -> 4C reacts with a 5C -> to form 6C and 3C.
What is the PPP?
The pentose phosphate pathway
What is the starting molecule of the PPP?
Pentose-6-phosphate
What is the irreversible step of the PPP?
The decarboxylation of pentose-6-phosphate
What enzyme catalyses link reaction?
pyruvate dehydrogenase
What cofactors are used in link reaction?
Thiamine Pyrophosphate, lipoic acid , FAD, Coenzyme A, NAD+
What does the Krebs Cycle generate?
ATP, NADH, and FADH2
What are anaplerotic mechanisms?
Mechanisms used to top-up the carbon count within the Krebs cycle, upon loss of intermediates for biosynthesis.
Example of an anaplerotic?
Carboxylation of Pyruvate into Oxaloacetate by pyruvate carboxylase.
What is the reaction equation of the carboxylation of pyruvate into oxaloacetate?
Pyruvate + CO2 + ATP + H2O -> Oxaloacetate + ADP.
Why do photosynthetic organisms do Krebs cycle backwards?
Because they incorporate CO2 into molecules
Can certain organisms not use the entire krebs cycle?
Yes
What is the benefit of an organism being able to use the Krebs cycle in both directions?
Allows for a balance of reduction and oxidation.
Why don’t anaerobic bacteria use the full Krebs cycle?
They don’t need it for the oxidative cycle or for metabolic precursors -> therefore run it backwards or split it into two arms.
How can fatty acids catabolism feed into the Krebs cycle?
the beta-oxidation of fatty acids.
What occurs in the beta-oxidation of fatty acids?
Faty acids are broken up into 2-carbon pieces forming acetyl Coa -> producing NADH and FADH2 for each 2-carbon piece.
What oxidation reaction occurs in beta-oxidation of fatty acids?
oxidises 2 carbon atoms furthest away from the acyl headgroup -. occurs repeatedly.
What enzyme is used for beta-oxidation of fatty acids?
Beta-carbon oxidase.
What is meant by Omega-3 fatty acid?
The alkene group is only 3 carbons from the end.
How is fatty acid biosynthesis the opposite of beta-oxidation?
Each addition produces a ketone group, that is then reduced in two stages.
What is the enzyme that catalyses the process of fatty acid synthesis?
Fatty acid synthase:
Features of fatty acid synthase?
Fatty acid synthase forms a dimeric protein that has 7 different enzyme functions in one polypeptide chain -> the fatty acid passes round the active sites in a cyclic process.
What occurs fatty acid synthesis to halt?
The fatty acid will become to large and be displaced from the enzyme.
Can ammonia be fixed by humans?
Yes, but not efficiently
Where is nitrogen sourced from in humans?
The consumption of plants and animals.
How is glutamate synthesised?
Alpha-ketogluatarate + Nh4+ -> Schiff base + H2O
Schiff base is reduced into glutamate by NAD(P)H
What is the coenzyme for amino acid biosynthesis?
Pyridoxal phosphate (B6)
How can nitrogen be passed between amino acids?
Transamination (glutamate can donate its amine to form alpha-ketoglutarate, a keto acid)
What are the essential amino acids:
Amino acids that are synthesised by the human body but are necessary for function.
Is making bases more or less complicated than aminoacids?
More complicated
Pyrimidine Nucleotide synthesis: Order
-Base assembled first -> ribose attached.
How is the pyrimidine ring formed in nucleotide synthesis?
The ring is formed by reacting aspartate with carbamoyl phosphate.
Where is carbamoyl phosphate sourced from in nucleotide synthesis?
A reaction between bicarbonate, NH3, and 2ATP.
Purine Nucleotide Synthesis:
-Ribose-5-phosphate forms 5-phosphorybosyl-1-pyrophosphate -> uses 2ATP
-> Intermediate atoms then added one at a time.
Why is ATP converted into AMP during the synthesis of purines?
This is because it’s a large energy change than that of the formation of pyrimidines -> therefore more energy released to ensure it occurs.
What is the committed step?
An irreversible step that commits to a reaction pathway.
What are the two ways metabolic pathways are usually controlled?
-Committed steps
-The end-product causing feedback inhibition of the first step
What molecule acts as an end-product allosteric inhibitor of glycolysis?
ATP -> inhibits the first step of glycolysis.
Why are lots of pathways often inhibited by ADP?
if ADP levels are high the cell likely doesn’t have the ATP required for the pathway.
How do most allosteric inhibitors work?
Most bind within the hydrophobic cleft -> influences how easy it is for active site to form.
How can allosteric inhibition be used as a switch?
It can downregulate one metabolic pathway, therefore upregulating alternative using the same starting molecule.
What are isozymes:
When there are more than one enzyme that do the same job; found in different conditions.
How are isozymes formed?
Often derived from the same gene but are differentially spliced (in eukaroytes)
Or are the same protein-coding domain but attached to differetn regulatory domains (in proakryotes)
Example of isozyme in E.coli?
Aspartokinase
What is the function of lactate dehydrogenase?
Enzyme that converts pyruvate into lactate.
Example of allosteric inhibition in eukaryotes:
aspartate transcarbamoylase, ATcase ->Nucleotide synthesis -> inhibited by the binding of CTP
Key feature of lactate dehydrogenase structure:
- has 4 subunits made of 2 different isozymes
- has H(heart) isozymes and M(muscle) isozymes
-H wants to convert lactate into pyruvate -> inhibited by pyruvate
-M wants to convert pyruvate into lactate -> inhibited by lactate
What is meant by the cumulative control of a single enzyme?
One single enzyme is inhibited independently by several products.
Example of enzyme controlled cumulatively:
Glutamine synthase (AMP, Histidine, Glycine, Alanine + more)
What is the effect of reversible phosphorylation on an enzyme?
Causes a regulated covalent modification -> Alters function
What is an example of an enzyme that is regulated by reversible phosphroylation?
Glycogen phosphorylase:
How does glycogen phosphorylase function?
-Removes glucose units from glycogen
-It’s activity is regulated by phosphorylation (enzyme that mediates this is regulated by glucagon)
What are the two methods of regulation by enzyme expression?
An end product downregulates expression of the first enzyme of a pathway.
Or a substrate acts to remove the repression of gene expression pathway needed to process it.
Example of regulation by enzyme expression? (prokaryotes)
Lac Operon.
Example of regulation by enzyme expression? (eukaryotes)
Trp Repressor -> trp operon encodes genes for the biosynthesis of tryptophan -> if there’s tryptophan already it binds to repressor and shuts down pathway.
Key difference between gene regulation and allosteric control:
Gene regulation is much slower taking hours, whereas allosteric can happen in milliseconds
What does metabolic flux analysis show?
That increasing the amount of regulatory enzyme doesn’t make that much difference -> overall rate much more dependent on the entire pathway.
What is the flux coefficient of an enzyme in a pathway?
How much each enzyme controls the entire pathway.
What are the three types of fibres in the Cytoskeleton?
Microtubules, Actin filaments, and intermediate filaments.
Difference between Sphingolipids and Phospholipids:
Sphingolipids contain Nh instead of O, often more saturated than phospholipids, and are mainly trans double bonds rather than cis.
Microtubules:
Forms pathways for intracellular transport - made from tubulin and span from nucleus to cell surface.
Intermediate filaments:
Made of fibrous proteins that crissscross the cell to hold it together to maintain structural integrity.
What are the 3 types of lipids in the membrane:
Phospholipids, Sphingolipids, and Sterols.
Actin filaments:
Made from actin -> runs around the inside of the cell membrane -> has multiple functions
what kind of linkage joins a lipid headgroup and tail?
An ester linkage.
How can membranes transition through phases?
By altering the external environment.
Why are phospholipid bilayers thinner than ordinary
The headgroups are closer together because the membrane is less ordered.
What can be added to a membrane to increase its width?
Cholesterol, by increasing the order of the membrane.
What is the benefit of membrane fluidity?
Allows for movement within the bilayer to occur.
Phosphatidylcholine:
Cylindrical phospholipid that packs well into flat bilayers.
Phosphatidylethanolamine:
Phospholipid with a smaller headgroup than fatty acid tail, causes curving of the bilayer.
What is the effect of different lipids of the surface membrane shape?
Different lipids can alter the shape/curvature of the membrane depending on their distribution between the two leaflets.
What is scaffolding?
Extracellular proteins interacting with the membrane, affecting its shape (e.g. clathrin)
How do membrane proteins affect cell membrane shape?
-Cylindrical membranes will keep membrane flat.
-Conical membranes will cause the membrane to curve.
-If protein inserts into single leaflet -> will increase its surface area -> causing curvature.
Are lipid anchored proteins in a fixed position?
No they will move with their anchor,
Why is the fluid mosaic model not completely correct?
-Membrane is less fluid
-Components localise into the membrane in specific membrane rafts -> rigid regions with cholesterol and sphingolipids.
What kind of membrane proteins tend not to be in membrane rafts?
Prenyl-anchored proteins
What is flipase?
An ATP-dependent enzyme which flips lipids between bilayer leaflets.
What is phase separation?
The driving force of the two leaflets having different lipid compositions -> creates different sub regions in the memrbane.
What does Atomic Force microscopy (AFM)show?
The height of different components and shows proteins embedded in the membrane
How does atomic force microscopy work?
laser reflects light on cantilever arm that taps the membrane surface.
What structures are proteins packaged into for transport?
Vesicles
How can the lateral mobility of proteins be measured?
FRAP (fluorscence) recovery after photobleaching.
What is the function of membrane rafts?
To bring membrane proteins together of keep them apart.
What modifications can be made to membrane rafts to organise the attachment of proteins?
Covalent modifications, e.g. prenylation and the attachment of GPI anchors.
Endocytosis:
The invagination of membrane to bring a membrane protein to the membrane or bound ligand molecule inside the cell. e.g. used to control number of aquaporins
What is often used to congregate lipid rafts together to form larger rafts?
Dimerisation.
What is the process of ligand-mediated endocytosis:
-> ligand binds to receptor -> leads to formation of raft -> proteins bind to the raft (Caveolin) -> cause inward curvature (invagination) -> Scaffolding proteins accentuate curvature -> Caveolae are pinched off at the top and moved into the cell.
What is patch clamping?
A technique used to investigate the conductance through ion channels
What does patch clamping show?
Membranes are insulated and charged particles can’t passively cross without channel/carriers.
What do fixed excited levels tell us about ion channels?
All channels have the same current when open.
What is the resting potential of the cell?
-60mV
At what voltage are channels open?
0Mv.
What is the Na+/K+ pump?
-ATP dependent pump which constantly pumps 3Na+ out of the cell and 2K+ in -> this is active because both ions are moving up their conc. gradients.
What are resting K+ channels:
-Open when cell is at rest
-not gated
-Allows K+ to leak out the cell all the time -. to make membrane potential negative
-Leakage occurs untill localised [K+] is equalised -> not major problem
What is a channel-inactivating segment?
A molecule that is attached to channels that reversibly plugs them to prevent over action.
What are the two types of nerve cells:
Motor neurones (Spinal cord to muscle)and Sensory neurones (tissue to spinal cord)
What occurs at a synpase?
An electrical signal is converted into a chemical signal at the synapse.
What is transient charge?
A temporary membrane potential
What is a nerve impulse?
A transient charge in the membrane potential.
How is a stronger nervous signal produced?
More rapid (increased freq) of action potentials.
What triggers the opening of voltage-gated Na+ channels?
The depolarisation of the membrane to -40mv.
What is the benefit of Na+ channel plughs?
They prevent the signal from travelling backwards.
What is the refractory period?
Time where the Na+ channels are plugged and cannot transmit an action potential.
What makes depolarisation of neurones an energy efficient process?
Not much work is needed to restore ion/concentration because only small change in concentrations (and therefore potential).
How does tetrodotoxin work as a poison in fugu?
blocks voltage-gated Na+ channels.
Myelin Sheathes:
- Insulate regions of nerve cells
-Lipid membranes insulates ions from cross across large regions
-Allows nerve impulses to travel faster (potentials can jump)
-Gaps even 100Micro m
What condition is caused by loss in myelin regions in the brain and spinal cord?
MS (multiple sclerosis)
The Nernst Equation:
2 Equations that calculate the energy produced by ion gradients across a membrane.
What is Equation 1 of Nernst?
Delta G = charge on ion x membrane potential (in V) x Faraday constant. -> relates membrane potential to free energy difference.
What is the faraday constant?
96485 J mol^-1 V^-1
What is the Equation 2 of Nernst:
membrane potential = (RT/charge on ion x Faraday) x ln ([out]/[in]) -> relates membrane potential to concentrations
What cells make up the myelin sheathe?
Schwann cells.
What equation is obtained by combining equation 1 and 2 in Nernst?
Delta G = RT x ln([out]/[in]) - zFE
What is the name for how the potential “jumps” between gaps in the myelin sheathe?
Saltatory conduction.
What is the basic problem with signalling?
The membrane is quite thick and proteins are fluid -> makes it difficult to transmit signals over large distances.
What is signalling vital response for?
Hormones, growth factors, infection, neural synapses, bacterial response to environment
What is signalling?
A method of how an extracellular change to the environment leads to a signal which will cause the cell the change its behaviour/expression
What are the common effects of signalling?
They often effect enzyme activation and gene expression.
What are the 4 major pathways for signals to enter a cell?
- Ligand binds to a receptor
-Ligand binds to GPCR
-Ligand binds to ligand-gated ion channel
-bind to intracellular receptor.
GPCR: Ras Protein how it works
Ras is an adaptor protein that binds to 3 GTP molecules, the third forms hydrogen bonds with two loops on the protein surface (One is threonine and other is glycine) -> When the GTP is hydrolysed -> causes switch regions to move inwards (off)
GPCR: How are changes to G-protein structure amplified?
The switches are associated with other proteins -> which amplify the change by moving aswell.
What enzymes turn G proteins on?
GEFs (guanine exchange factors)
Receptor-linked kinases are an example of which signalling pathway?
Ligand binding to a receptor.
What do receptor linked kinases often control?
Transcription -> therefore defects can lead to cancer.
How do Receptor tyrosine kinases work?
Use dimerisation mechanism to transduce signal.
How does the dimerisation of receptor tyrosine kinases function?
Each receptor has an inactive kinase, when pulled together by the substrate -> kinases activate eachother (via autophosphorylation) -> the kinases can then activate secondary messengers.
How do kinases work?
-> kinases have 2 domains -> N-terminal domain binds to ATP and C-terminal domain binds to the substrate -> upon binding to both the domains become closer and the substrate is phosphorylated.
How are kinases activated?
Kinase binding to protein is weak -> upon binding the C-terminal domain to a phosphate (via the tyrosine residue) loop becomes more structured -> allowing for stronger binding to substrate. -> activating the kinase.
What is the function of modular adaptor molecules in cell signalling?
They connect specific phosphorylated receptors to a more general signalling molecule.
Grb2 Sos mechanism: Adaptor signalling
Grb2 -> has two SH3 domains which recognise polyproline helices (binds SOS)
Has SH2 domain that binds to phosphotyrosine
SOS has GEF domain that activates G-protein
What does Ras activate?
Activates a kinase cascade Ras -> MEK -> ERK -> phosphorylates TFs.
What is the general kinase cascade?
MAPKKK -> MAPKK -> MAPK -> phosphorylates TFs.
GPCR signalling pathway summary:
Signal binds to GPCR -> activates G-protein -> activates adenylyl cyclase -> synthesis cAMP -> cAMP protein kinase A -> activation of protein kinase A -> subunits to diffuse into nucleus -> phosphorylate a CREB -> recruits CBP -> activates transcription.
What is a CREB?
cAMP-response element binding protein
GaS: GPCR
Stimulatory GCPR
GaI: GPCR
inibitory -> inhibits cAMP pathway
GaT: GPCR
involved in vision -> stimulates phosphodiesterase 6 -> breaks down cGMP
GaQ: GPCR
Stimulates phospholipase Cbeta -> splits into PIP2, DAG, and IP2
Inositol phosphates:
Used to make signal and are released upon lipase activity cleaves internal lipid bilayer.
Examples of diseases that affect GPCR signalling:
Cholera -> G-protein not deactivated -> over excretion of Cl- and water
Whooping cough -> block GaI -> over expression of adenylyl cyclase -> excess cAMP.
What is the role of arrestin?
Performs mechanism for turning off GPCR signalling by lysosome action.
Outline arrestin-mediated signalling by GPCRs:
GRK phosphorylates receptor -> receptor binds to arrestin -> induces invagination of the protein (via AP2 clathrin) -> forms endosome -> then there are 3 different outcomes.
What are the 3 outcomes of arrestin-mediated signalling by GPCRs?
-Internalised receptor
-Receptor hydrolysed
-Receptor recycled.
What is NAChr?
Nicotinic acetyl choline receptor.
What causes ion channels to open?
The rotation of alpha helices (like an iris)
Ion channel properties:
-Rapid
-Reversing signal requires removal of all the ions -> requires a lot of energy.
What pathway is best suited for specificity and regulation?
Tyrosine Kinases
Signalling by hydrophobic ligands:
-> usually steroids
-> Receptors are in the cytoplasm
-> Receptors have 3 defined domains, DBD, AD, and LBD
-> Upon ligand binding conformationcal change occurs -> releases receptor from inhibitor + activate the receptor -. travels to DNA and triggers transcription
How do proteins associate with the membrane?
-Integral Membrane Proteins: Associate with hydrophobic environment
-Lipid-anchored Proteins: Associate with lipid anchors on the membrane surface
-Peripheral Membrane Proteins: associate with other membrane proteins
What interactions occur between peripheral membrane proteins and the membrane?
electrostatic forces and hydrogen-bonding with head groups -> polar interactions that are easily disrupted.
What is a hydropathy plot?
A window of 19 residues used to calculate the hydropathy of the polypeptide stretch.
What is the hydropathy index threshold for a transmembrane helix region?
+84
What residue is affected by acylation?
Glycine -> N-terminal methionine is removed -> forms amid bond with C14 carboxylic acid
What is the effect of acylation on membrane proteins?
Prevents the proteins from moving back into the cytoplasm.
What residue is affected by prenylation?
Cysteine -> unsaturated anchor binds to C-terminus of the protein -> forms thioester link
Structure of porins:
-Beta strands form large beta sheet -> rolled into a cylinder and pushed into membrane to form a tunnel
-Each strand is arranged in an anti-parallel arrangement.
What is the role of aromatic side chains on porins?
To dampen the cylinder at the point between different environments.
What type of interaction is important in making protein arrangements energetically feasible?
Hydrogen bonding
Where are porins found?
ONLY on the outer-membrane of bacteria -> to prevent protons from entering the cell interior and inhibiting ATP synthesis.
How does the polarity of an amino acid affect its hydrophobicity?
More positive transfer free energy = less hydrophilic/
More negative = hydrophobic.
Why can’t hydropathy plots be used for porins?
Because porins aren’t alpha-helical
Phenylalanine:
residue with single aromatic ring
Tryptophan:
Residue with two aromatic rings.
What is the width of Bacteriorhodopsin?
45 Angstroms
Where does the structure of bacteriorhodopsin derive from?
Archaea
Function of Bacteriorhodopsin:
To use light energy to transport protons from inside to outside the cell, generating a proton gradient to drive ATP synthesis.
What type of proteins are most membrane proteins (In regards to their secondary structure)?
Alpha helical
What do permeabilities correlate with?
Partition coefficients
What is the membrane permeability equation?
Permeability = (Diffusion Coefficient within membrane x water-membrane partition coefficient)/Membrane thickness
What factor other the partition coefficients is affects the ability of molecules to spontaneously cross the membrane?
The relative concentrations of the molecule across the membrane.
What is a key example of a channel protein that allows for passive transport?
Aquaporins
What are the key 3 features shared by channel proteins:
-Selectivity
-Filter
-Gate (optional)
What are the shared components of Channel proteins?
->Selective tunnel
-> Hydrophobic interface (lots of phenylalanine) to protect pore from hydrophobic interior
-> Vestibule (cavity) on either side to collect the substrate.
->Gate (acts as a switch)
Aquaporins:
Facilitate the movement of many more water molecules across the membrane in a given time.
-e.g. in the kidney for rapid diffusion back into the bloodstream after filtration
How fast are aquaporins?
Operate near the diffusion limit.
What are the 3 types of transport processes?
-Uniporters (typical channels + Passive)
-Symporters (Couple of two molecules in the same direction + Active)
-Antiporters (Couple of two molecules in the diff directions + Active)
How do ATP pumps work?
Use chemical energy (ATP) to undergo large conformational changes to the pumps structure, allowing for the movement of molecules up the conc. grad.
How does facilitated diffusion occur?
Facilitate diffusion down gradient by undergoing smaller conformational changes induced by the energy of binding with the molecule.
What does the Jardetzky model depict?
A simple allosteric model for active carriers.
What are the components of the Jardetzky model?
-An interior binding cavity that binds to transported species (high affinity)
-Channel protein can assume two different configurations -> provides alternating access.
-An energy input induces a conformational change that alternates access and decrease affinity of the interior binding cavity.
Lac permease mechanism:
Ley amino-acids (aspartates and glutamates) in aq environment and _ve charged -> proton will bind to acidic amino acid -> causes change in binding site of lactose -> conformational change causes eversion -> loss of affinity of lactose -> lactose and H+ released -> lac permease returns to resting state.
What is a word that can be used to describe a change between conformations in alternative access?
eversion
What is the source of energy for the action of lac permease?
The proton gradient.
What kind of active transport protein is lac permease?
A symporter (of lactose and H+)
What family of proteins is lac permease part of?
Major facilitator family
What do kinks in alpha helices suggest?
Flexibility of the protein.
What does PSI reduce?
Ferredoxin
What are the pigments in the reaction centre of PSI?
Chlorophylls (aA1 + aB2) + (aA2 +aB2) + Phylloquinone (QKA + QKB) and 4Fe4S clusters
What resolves the charge at the special pair of electrons in PSI?
Plastocyanin
Where is the reduced plastocyanin sourced from in PSI?
Cytochrome b6f.
Structure of lac permease:
-12 transmembrane helices -> 2 separate 6 helices domains (pseudosymmetry)
How was the structure of Lac permease found?
X-ray crystallography of a mutant locked in a single conformation.
What energy does Bacteriorhodopsin use to drive its active transport?
Light energy
How does the Light ensure the directionality of bacteriorhodopsin?
Input of light closes the gate, preventing the backflow of electrons .
What bacteria is bacteriorhodopsin found in?
halophilic bacterium in brine lakes -> Halobacterium salinarum
What responsible for bacteriorhodopsin’s colour?
Purple colour comes from covalently attached chromophore.
What are the the important amino acids in bacteriorhodopsin?
D85 (aspartate -> near retinal), D96 (aspartate -> near cytoplasm), and Lys216 (schiff base)
How many helices surround the retinal in bacteriorhodopsin?
7 helices
Function of D85 in bacteriorhodopsin:
D85 acts as a counterion to stabilise the proton bound to lysine.
What is the function of D96 in bacteriorhodopsin?
Forms a hydrophobic environment -> protonates because negative charge isn’t favourable in hydrophobic environment.
What does the absorption of light cause in bacteriorhodopsin?
Isomerisation at C13 -> causes system to strain ->pushing against the protein.
What are the stages of H+ pumping by bacteriorhodopsin?
-Light absorbed by retinal -> isomerisation -> allows access of cytoplasmic side to retinal -> lysine donates electron to D85 (is then released) and D96 donates electron to lysine-> proton form binds to D96 system restored.
How does gating occur in bacteriorhodopsin:
(M stage)
Upon light absorption Helix F has a large movement outwards at the cytoplasmic end -> allows cytoplasm to fill half the channel -> allows for decrease D96 pKa -> dissociates H+ to retinal.
Retinal strain relaxes-> helix F closes -> makes half channel hydrophobic -> allows for protonates D96
Why are K+ ions 100x more permeable in K+ channels than Na+?
Despite Na+ being smaller the energetic of its hydration require higher energy input to remove water
What is the desolvation energy cost?
The energy cost of removing an ion from associated water molecules.
What is KcsA?
A potassium ion channel found in streptomyces
KcsA structure:
Tetrameric channel protein: Each subunit has 2 transmembrane helixes connected by a pore helix with a signature sequence.
has a selectivity filter formed by 4 pore helices.
What is the purpose of the signature sequence on the pore helix of a pore protein (KcsA):
Carbonyl groups on the main chain in the pore lumen which provide selectivity by electronegativity
Structure of KcsA subunits: Outer helix
Hydrophobic helix that forms outersurface of the pore
Structure of KcsA subunits: Inner helix
Lines the vestibule and pore (helical pore has dipole that propels +ve forward)
What force between ions helps aid the movement of K+ through KcsA?
Electrostatic repulsion
What is KvaP?
Voltage gated K+ channel
What links the pore domain and voltage domain in KvaP?
Linker domains
Why does KvaP have 4 more helices than KcsA?
They are critical for the gating mechanism (contain the sensor for voltage change)
KvaP: N-terminal Voltage Sensor
Moves up and down depending on the membrane potential (voltage) -> acts like a level mechanism which will push and pull by transient movement on inner helices in pore domain.
KvaP: Subunits and purpose:
S5 and S6 -> analogues of the inner and outer helices of KcsA.
S1-4 -> form membrane embedded voltage sensor.
KvaP: When the sensor are up is the channel open or closed?
Open
What is the effect of glycine and prolines on transmembrane alpha helices?
They create a break in the helix -> indicating a hinge region.
Where was rhodopsin initially harvested from?
Cows and squid
How many Transmembrane helices does rhodopsin have?
7
Is rhodopsin and bacteriorhodopsin releated?
No, but share similar structure.
Role of retinal in rhodopsin?
Acts similar to a ligand on other GPCRs.
Where is beta adrenergic receptor’s binding site?
The extracellular side
What is the role of ligand binding on beta adrenergic receptors?
Energy of binding of ligand to binding site -> induces conformational change -> communicated to cytoplasmic side of receptor -> recognised by G-protein.
What signalling cascade is rhodopsin involved in?
Sight.
What membrane protein is activated by the activated alpha G protein subunit in Adrenaline receptors?
Adenylate cyclase
Role of Adenylate Cyclase:
Converts ATP into cAMP for signalling cascade (activates protein kinase)
At which steps of GPCR mechanisms does amplification occur?
For each ligand, multiple G proteins are activated -> binding of each results in multiple cAMP molecules.
What happens to G protein when activated by GPCRs?
GDP dissociates and it replaced with GTP -> mediated by GEF.
What are components of a heterotrimeric G protein?
Alpha, beta, and gamma subunits
What is the effect on G alpha when bound to GTP?
It has a decreased affinity of the receptor and dissociates from the receptor and beta and gamma -> then associates with downstream proteins (e.g. adenylate cyclase)
Adenylate cyclase structure:
12 transmembrane alpha helices and two large cytoplasmic extramembraneous domains.
How do G-proteins deactivate:
-They can spontaneously reset through GTP hydrolysis via intrinsic GTPase activity
-> will the reassociate with gamma and beta subunits and form inactive heterotrimeric G-protein
How does a GPCR receptor deactivate?
-The ligand will dissociate -> returning the receptor to its inactive state
- Or kinase will phosphorylate serines and threonines on the c-terminal end -> recruit beta-arrestin -> blocking recruitment of G-protein.
What key group do threonines and serines have?
Hydroxyl group
Where is the cis bond in rhodopsin’s retinal’s polyene tail?
11-cis carbon
Where is the retinal ring in rhodopsin?
In the binding cavity
What is the change that occurs in retinal of rhodopsin upon light absorption?
changes from 11-cis to all-trans .
What is the mechanism by which rhosopsin conformational change occurs?
Upon isomerisation helix 6 and 3 are separated (possibly as a result of proton transfer from Schiff base to glu113) -> helix 6 relaxes and goes outwards, at the other end this causes conformation at the transducing complex.
Visual signal transduction system post Rhodopsin:
G alpha subunit dissociates from rhodopsin and regulate phosphodiesterase -> decreases cGMP -> reduces binding to cGMP-gated ion channels -> prevents entry of cations -> membrane becomes hyperpolarised and neurotransmitters released.
How is rhodopsin deactivated?
-Phosphorylation and recruitment of arrestin
-Or dissociation of retinal
Why isn’t light used to observe molecules?
The wavelengths of visible light is too large to observe protein and biological molecules.
What was the original method of observing proteins?
X-ray scattering -> scattering pattern and density analysed computationally to reverse engineer structure.
X-ray crystallography issues with protein crystals:
Protein crystals are very fragile due to weakness of intermolecular interactions.
Why were protein crystals used in X-ray crystallography?
They provided lots of copies and could be used despite the lack of lenses.
What is the unit cell?
3D repeating unit that is the lattice structure repeated throughout the crystal.
Why must the protein solution be very saturated?
To make sure the protein leaves as a crystal rather than a precipitate.
What is X-ray crystallography resolution dependent on?
The quality of the crystals.
Difficulties of crystallising proteins:
-Disruption of bilayers
-Aggregation of proteins occurs in water
->Detergent use must be very precise.
Detergents:
Water-soluble surfactants that form micelles that separate hydrophobic species from the water.
What is the resolution of recent CryoEM:
3 Angstroms
Process of Cryo-EM:
Apply sample to EM grid -> blotted -> plunged into liquid ethane -> transferred to EM microscope -> at random orientations -> can build 3D shape.
What are the benefits of Cryo-EM:
-No phase problems and pictures can be taken
Why don’t crystals form in Cryo-EM?
the protein freezes so quickly in the ethane.
SecYEG complex:
Membrane complex that acts as a pore that allows for the active transport of proteins out of the cytoplasm into the periplasm.
What is an SRP?
A signal recognition particle
What is the purpose of the FtsY:
Binds to SRPs (docking + hydrolysis of GTP) will trigger the ribosome forms a complex SecYEG pore -> protein will then be translated and fed through the pore simultaneously.
What are the three ways of translocating a protein:
Co-translational translocation, Post-translational translocation (BiP or SecA ATPAse)
What is the role of SecA:
->recognises unfolded protein -> binds together by polypeptide linking domain -> clamp around the protein and bringing it to the SecY complex -> will act as a seal whilst the ppeptide is fed through the pore -> cleaved after translocation is complete.
Stages of Co-translational translocation:
Free ribosomal subunits assemble at a signal peptide -> mRNA is translated across the ribosome -> synthesised polypeptide is fed through translocator -> upon completion signal peptidase cleaves the signal peptide off the polypeptide.
Which translocation mechanisms use the Sec61 complex?
Co-translational translocation and BiP
What is the eukaryotic analogue of FtsY?
SRP receptor
What are SAIs?
Signal Anchor I proteins
What do SAI proteins indicate?
Indicate TM segments transfer into lipid during co-translational translocation.
What is topogenesis:
The process by which proteins are integrated into the membrane.
What are the 3 types of signals that initiate topogenesis in membrane proteins:
- Cleavable proteins
- Signal-anchor
- Reversible-signal anchors
Components of translocators:
-Hinge and Seam
-Plug
-Lipid-bilayer
What factors effect the orientation of a membrane protein:
The number of positive and negative charges either side of the signal peptide + large globular domains don’t easily pass through the translocator + the hydrophobicity of the N-terminal domain.
What occurs in single pass membrane protein integration:
Translocator binds to start-transfer sequence -> translocation occurs -> until stop-transfer sequence -> start-transfer seq. cleaved.
What occurs in double pass membrane protein integration:
Similar to single pass except there’s polypeptide before the start-transfer seq. -> start-seq. is not cleaved -> forms loop.
Example of modification of membrane proteins:
Glycosylation, phosphorylation, etc.
