Membrane proteins

Question 1

Roles of membrane proteins

Answer 1

Transporters, channels, anchors (like integrins), receptors and enzymes

Question 2

Classification of membrane proteins by nature of association with membrane:

Answer 2

Integral proteins interact with hydrophobic hydrocarbon tails, mostly membrane spanning.

Peripheral proteins **attached to membrane by phospholipid head groups. **or to integral proteins

Question 3

What kind of membrane protein is bacteriarhodopsin an example of?

Answer 3

Membrane spanning alpha helices.

(7 transmembrane alpha helices, common number

forms a channel for H+)

(contains retinal, light activated conformational change pumps H+ out across membrane)

Question 4

Rough width of lipid bilayer?

how many hydrophobic aa residues required to form a window that can make a membrane spanning alpha helix?

Answer 4

30 angstroms (10^-10) [3 nanometres]

20 hydrophobic amino acids can form a window.

Question 5

What is a hydropathy plot?

Answer 5

Lists amount of energy required to move an aa residue from membrane to water. (free energy of transfer)

Hydrophobic amino acids have +ve values. (therefore give a peak on the plot)

Hydrophilic amino acids happily move to water and therefore have negative values.

Question 6

What is a porin?

Answer 6

Porin is a transmembrane beta sheet structure. (beta barrel)

Found in bacteria and mitochondria.

(C and N terminals on same side?)

Beta barrels have outer hydrophobic side and inner (pore forming) hydrophilic side.

Question 7

Structure of a beta barrel?

Answer 7

Antiparallel beta strands form a single beta sheet (hydrogen bonded to each other)

Curls ups into hollow pore.

Alternating hydrophobic and hydrophilic residues along each strand creates hydrophobic outer side, and hydrophilic inside.

N terminal and C terminal end up close to each other. (contrasting most transmembrane alpha helices)

Question 8

What is a monotopic protein?

Answer 8

An integral membrane protein that is not membrane spanning!

Only part of its structure embedded in membrane.

E.g.prostaglandin H2 synthase and AOX

Question 9

Prostaglandin H2 synthase?

Answer 9

A monotopic protein which Converts arachidonic acid into prostaglandin H2.

(Which promotes inflammation and helps regulate gastric acid secretion.)

[Mostly alpha helical homodimer, held in membrane by alpha helices with hydrophobic side chains.]

Aka COX (cycloxygenase) (as inhibited by aspirin)

Question 10

Role of hydrophobic channel of Prostaglandin H2 synthase (COX1)?

Answer 10

alpha helical channel of this monotopic protein extends into membrane, provides hydrophobic route for substrate from membrane to active site within protein.

Within this channel is the Serine530 residue bound by aspirin/ibuprofen to inhibit COX activity.

Question 11

What are the 2 dehydrogenases of the ETC?

Answer 11

Complex I (NADH dehydrogenase) (also pumps protons)

Complex II (Succinate dehydrogenase)

Both reduce Ubiqinone (co-enzyme Q10) by transferring electrons to it.

Question 12

Complex III?

Answer 12

Cytochrome bc1 complex.

Transfers electrons from Co-enzyme Q10, ubiquinone to Cytochrome C (cyt C)

Pumps 4 protons across inner mitochondrial membrane.

Question 13

What is collisional interaction?

Answer 13

Random movement of respiratory chain complexes allowing them to interact with each other.

(higher numbers of UQ and cyt-c facilitate increased speed of this interaction)

Question 14

Complex IV?

Answer 14

Cytochrome c oxidase. (or cyt-aa₃)

Oxidises (removes electrons from) Cyt-c

Reduces 1/2O₂to H₂O!

[13 subunits, functions as a homodimer.]

(Uses 4H+ but only pumps 2 out, uses 2 protons to make water molecule.)

Question 15

What is the Chemiosmotic hypothesis?

Answer 15

A theory (determined by Mitchell in 1961) linking ATP synthesis to generation of PMF (electrochemical grad) across inner mitochondrial membrane, by electron transport using energy from NADH and FADH₂ generated from catabolising energy rich substrates such as glucose.

It requires 4 basic tenets.

Question 16

4 Basic tenets of chemiosmotic theory?

Answer 16

Ion impermeable inner mitochondrial membrane.

Proton pumping ETC

H+ or OH- linked exchange diffusion system

Reversible H+ translocating ATP synthase

Question 17

How are protons transported across the inner mitochondrial membrane? (2 ways)

Answer 17

1) By poorly understood **active transport by complexes. **(I,III,IV)

2) Or transported by ubiquinone:

QH₂ formation (reduction by Complex I or II) accepts 2 protons from matrix side.

QH₂oxidation by cytochrome bc1 (complex III) releases 2 protons into intermembrane space.

Question 18

What is a cytochrome?

(e.g. a, b, or c)

Answer 18

A hemeprotein. Contains Fe2+ in a porphyrin ring.

Question 19

Components of proton motive force?

Answer 19

Electrical gradient (more positive outside): largest driver (190mV, ΔΨ)

Chemical gradient (more H+ outside, more acidic outside): ΔpH of 60mV

Total 250mV = Δp = PMF

Question 20

Metals in Cytaa₃? (complex IV) (cytochrome c oxidase)

Forming the 2 binuclear centres

Answer 20

2 Fe2+ haem groups (a and a₃)

3 copper ions

(2 CuAs form first binuclear centre)

(CuB binds to a₃iron to form second binuclear centre)

Question 21

Transfer of electrons within complex IV (cytaa₃) (cytochrome c oxidase)

Answer 21

1) Electrons from cyt-c to CuA binuclear centre.
2) To cytochrome a
3) To cyta₃- CuB centre
4) to oxygen O₂

Cyt-c - CuA - Cyt-a - cyt-a₃-CuB - O₂

Question 22

Core structure of Complex IV?

and significance of hydrophobic core?

Answer 22

Transmembrane alpha helices, subunits 2 and 3 wrapped around subunit 1.

Subunit 1 has hydrophobic core in which oxygen reduced one electron at a time!

Hydrophobic core with CuB and cyta₃binuclear centre stabilises oxygen radical, preventing its release. (O₂ bonded to a₃iron)

Question 23

How does complex IV (cytochrome c oxidase) transport protons across the membrane?

Answer 23

Movement of electrons (down energy states) provides energy for a conformational change that moves bound protons across protein to intermembrane side.

High proton affinity state accepts protons from matrix, conformational change moves proton across membrane and releases it. (possibly via chain of acidic residues like glutamic acid, E, on alpha helices)

Question 24

Function of alternative oxidase? AOX

Answer 24

Alternative oxidator of ubiquinone found in plants and fungi and protists. And some human pathogens!

Reduces oxygen to water, used as oxygen scavenger in parasites. But it does NOT pump protons. (so smaller/no PMF made [complex I still pumps a few])

Stress protein in plants

AOX is faster at reducing oxygen than whole pathway.

Question 25

How do some plants generate heat?

Answer 25

Through use of alternative oxidase, fast respiration without ATP generation.

Question 26

Trypanosomiasis pathophysiology?

Answer 26

Sleeping sickness in human, Nagana in livestock: trypanosome parasite transmitted by tsetse fly.

Trypanosoma brucei replicates in bloodstream, enters nervous system, burns all energy using AOX so none for host!

Question 27

Problems with treatment of Sleeping sickness (trypanosomiasis)

Answer 27

Large immunocompromised population in africa (AIDs related)

Drugs mostly obsolete and difficult to administer (e.g. IM and IV injections)

Mechanisms of action unclear.

Question 28

State of the respiratory chain in Trypanosome life stages? (insect stage vs bloodstream stage)

Answer 28

Full complement of respiratory chain in insect stage.

AOX upregulated in bloodstream stage, everything else downregulated!

Takes in hosts ATP and Glucose (burns it)!

Glycolytic pathways upregulated hugely in host.

Question 29

Cryptosporidium parvum, what is it?

Answer 29

A parasitic protozoan.

causes cryptosporidiosis, characterised by profuse diarrhoea, of particular danger to immunocompromised patients.

contains AOX.

Question 30

What is Blastocystis hominis?

Answer 30

AOX containing common protozoal parasite that causes diarrhoea in humans,

of concern for immunocompromised patients!

Question 31

Known characteristics of AOX?

Answer 31

Homodimer. Monotopic. Di-iron binding protein. (2 iron atoms coordinated by glutamate residues, E) with hydrophobic channels between membrane anchoring alpha helices 1 and 4 for UQ leading to di-irons

No cytochromes/chromophores present. (no Fe-S or flavins)

Reduces Oxygen to water, not hydrogen peroxide, so complex stepwise reduction.

Question 32

F₁Subunits, which has the ATPase catalytic site?

alpha (3)

beta (3)

gamma (1)

delta (1)

epsilon (1)

Answer 32

The 3 beta subunits each have one ATPase catalytic site

(alphas: regulatory, gamma: shaft, delta: part of peripheral stalk prevents movement, epsilon: inhibitor of reverse rotation)

Question 33

How do ß subunits (of F1) change conformational states?

Answer 33

By interaction with rotating gamma stalk. (one arm of central stalk is bent, and this interacts)

Interaction with stalk induces ß-empty state, nothing bound

Question 34

Structure of ATP synthase F₀ subunit?

Answer 34

A proton pore with 3 subunits

a,b₂,c_8-15 (c numbers vary by species)

c subunits each have 2 small very hydrophobic** alpha helices arranged in concentric rings**. (1 inner, 1 outer helix) (perpendicular to plane of membrane)

Inner ring made of amino terminal helices,

Outer ring made of carboxy terminal helices

(small matrix-side loop inbetween)

Question 35

Peripheral stalk of ATP synthase?

(bacterial)

Answer 35

Stabilises catalytic “head piece” against rotation of gamma stalk.

Consists of 2 long b (F0) subunits, and 1 delta (F1) subunit. (in bacterial ATP synthase, more complicated in mitochondrial)

Question 36

How to protons move through the F₀subunit?

Answer 36

Entry through half channel in subunit A,

Loaded onto an aspartate residue in a C subunit.

This rotates whole C ring.

Proton released once its C subunit rotates around the ring (driven by loading of further protons), and reaches exit half channel in subunit A.

Question 37

How did some japanese guys prove C ring of F0 rotates?

Answer 37

Removed whole ATP synthase from membrane.

Immobilised F1 (stuck its histidine residues to nickel)

Attached actin filament to c subunit.

120deg per ATP (and 3 protons)

Question 38

What are the 3 conformational states of F1 beta subunits?

(binding change mechanism)

Answer 38

LOT (bifurcated gamma stalk going clockwise around, but each subunit goes TOL over time)

Loosely bound ADP+Pi (ß-ADP)

Open (ß-empty) (interacting with gamma stalk)

Tightly bound ATP (ß-ATP)

Question 39

How does AOX reduce oxygen to water?

Answer 39

Binds O₂to one of its irons (in di-iron)

Transfers single electrons by generating amino acid radicals: First Tyrosine, then Tryptophan

1 QH₂(ubiquinol) reduces O₂further to H₂O.

Then another ubiquinol quenches the aa radicals generated earlier!

Question 40

What is cytochrome b₆-f in plants?

Answer 40

cytochrome b6-f is similar to cytochrome bc₁(complex III) in animal mitochondria.

Catalyses the transfer of electrons from photosystem II to photosystem I (via plastoquinone to plastocyanin)

Question 41

What provides the electrons for the plant (thylakoid) respiratory chain?

Answer 41

Photosystem II, specifically charge separation at the special pair of chorophylls in the reaction centre.

(passed to plastoquinone, to b6f, to plastocyanin to PS-I)

Question 42

Roles of Photosystem II?

Answer 42

Split water using water splitting enzyme with manganese ions,

to produce oxygen, protons and electrons.

Contains special pair of chlorophylls (with magnesium centres) that transfer electrons (one per photon absorbed) to plastoquinone. (manganese ions from water splitting enzyme then replace electrons.)

Question 43

Features of bacterial photosynthetic reaction centre?

transfer route of electrons?

Answer 43

Complex of 3 polypeptides.

Special pair Chlorophyll dimer absorbs photons.
Passes electrons down one side of dimer structure.

To accessory chlorophyll

To bacteriopheophytin

Finally to a quinone.

In conditions of excess light, the other side could act as a return pathway for electrons! (via iron atom)

Question 44

Role of light harvesting complex 2? (LH2s)

Answer 44

Ring structures in dimers,

Absorb light energy and transfer it to LH1s.

LH1s have reaction centres.

Question 45

Why is the LH1, RC, PufX dimer S shaped?

Answer 45

To allow gaps for the diffusion of plastoquinone out.

Question 46

What other protein is required for the LH1-RC “S-shaped” dimers to form?

Answer 46

1 PufX per monomer, small transmembrane protein

2 PufX monomers dimerise at their n-terminal regions

Question 47

How do photosynthetic chromatophores occur?

Answer 47

Curved structure of LH1-RC-PufX dimer complexes causes large areas of membrane invagination that function as cellular organelles.

(in photosynthetic bacteria)