Lecture 3- Membrane Proteins

Question 1

functional evidence for proteins in membrane

Answer 1

Channels and transporters - facilitated diffusion - ion gradients - specificity of cell responses

Question 2

biochemical evidence for protein in membrane

Answer 2

• membrane fractionation and gel electrophoresis - freeze fracture

Question 3

example of cell fractionation and feel electrophoresis of erythocytre membrane

Answer 3

(no organelles) 1. breaking up of cells e.g. with detergents 2. centrifuge 3. electrophoresis on SDS-page

Question 4

electrophoresis separates proteins based on

Answer 4

size and charge - smallest migrate furthest towards positive electrode

Question 5

freeze fracture is a technique

Answer 5

, a specimen is frozen rapidly and cracked on a plane through the tissue. This fracture occurs along weak portions of the tissue such as membranes or surfaces of organelles.

Question 6

freeze fracture process

Answer 6

1. Take cell and freeze- Locks cell membrane in position 2. Use sharp knife to fracture ice crystal 3. Fracture occurs where there is points of weakness- between bilayer 4. Can look at both sides of the phospholipid bilayer 5. Preparation can then be looked at under TEM

Question 7

freezing cell

Answer 7

locks cell membrane in position

Question 8

freeze fracture shows how

Answer 8

dense the membrane is with protein (60%)

Question 9

mobility of proteins (3)

Answer 9

-Conformational change - Rotational- fluid lipid environment -Laterally- like phospholipids (nearly as fast)

Question 10

why can’t proteins flip flop

Answer 10

due to thermodynamics- would break membrane

Question 11

when protein density within membrane is high what forms

Answer 11

aggregates of proteins are called RAFTs

Question 12

RAFTs can

Answer 12

restrict protein motion

Question 13

proteins aggregates found

Answer 13

in cholesterol rich regions

Question 14

tethering of cells to basement membrane/ ECM/ cytoskeleton etc can

Answer 14

stop proteins move in and out of cells

Question 15

lipid associated effects e.g. cholesterol rich regions are more packed

Answer 15

restrict protein mobility

Question 16

restriciton of membrane mobility (2)

Answer 16

• RAFTs - Tethering - lipid associated effects

Question 17

type of membrane proteins

Answer 17

1) Peripheral 2) Integral

Question 18

peripheral proteins

Answer 18

Bound to the surface of the cell membrane- not studded in membrane

Question 19

peripheral proteins are bound to the membrane by

Answer 19

electrostatic and hydrogen bond ionteractions

Question 20

how can peripheral proteins be removed

Answer 20

by changing pH or ion solution

Question 21

integral proteins

Answer 21

• Interact extensively with hydrophobic domains of the lipid bilayer - Found within the membrane

Question 22

how can integral proteins be removed

Answer 22

i. Cannot be removed by manipulation of pH and ionic solution ii. Are removed by detergents that compete for non-polar interactions

Question 23

hydrophillic part of protein will always be

Answer 23

on the outside of the lipid membrane

Question 24

hydrophobic part of the protein will always be found

Answer 24

in the inside of the bilayer

Question 25

transmembrane polypeptide strucuture

Answer 25

often alpha helical - R groups of amino cid residues I transmembrane are largely hydrophobic

Question 26

what can be used to can translate proteins sequence of polypeptide and score the amino acids for Hydrophilicity or hydrophobicity

Answer 26

Hypdropathy plots

Question 27

glycoproteins membrane topology

Answer 27

• End terminal of protein will be in the cytoplasm • Oligosaccharide will be on the outside of the cell

Question 28

N terminus on the

Answer 28

outside fo the cell e.g. where the ligand binds - oligosaccharide

Question 29

C terminus

Answer 29

end terminal of protein will be in cytoplasm

Question 30

normal erythrocyte cytoskeleton influences structure

Answer 30

makes it biconcave- large surface area and can squeeze through capillaries

Question 31

cytokseleotn maintains

Answer 31

RBC structure- spectrin lattice

Question 32

what can be used to visual proteins in the erythrocyte cytoskeleton

Answer 32

electrophoresis - Peripheral membranes washed off (salt wash) - Desired proteins (spectrin) can be visualised on electron microscope

Question 33

most important protein involved in the erythrocyte cytoskeleton

Answer 33

Spectrin - maintains RBC integrity

Question 34

how is the spectrum lattice adhered to the cell membrane

Answer 34

- runs parallel to the cell membrane - Ankyrin binds spectrin to Band 3 (integral protein in cell membrane)

Question 35

if something goes wrong with the formation of the spectrum lattice

Answer 35

- weakness in cytoskeleton will occur- reusing structural integrity o the Roc --\> these RBC will be damaged in blood vessels and destroyed by the body leading to anaemia

Question 36

Weakness in spectrin lattice leafs to two types of

Answer 36

haemolytic anaemia

Question 37

two types of haemolytic anaemia

Answer 37

- hereditary Spherocytosis - hereditary Elliptocytosis

Question 38

hereditary Spherocytosis

Answer 38

- Spectrin depleted by 40-50% - Erythrocytes round up- blow up - Less resistant to lysis - Cleared by spleen

Question 39

hereditary Elliptocytosis

Answer 39

- Defect in spectrin molecule - Unable to form heterotetramers - Fragile elliptoid cells- rugby ball shape

Question 40

secreted protein synthesis e.g. insulin

Answer 40

1) mRNA produced in the nucleus moves to the cytoplasm where it forms a complex with the two subunits of a ribosome 2) The mRNA begins translating the mRNA 3) The first part of translated is the signal sequence 4) The signal sequence recruits signal recognition particle (SRP), which halts translation. 5) The SRP- ribosome complex then docks at a receptor located on the ER (forming rough ER) 6) Translation is re-initiated and the polypeptide chain continues to grow via a transport channel into the lumen of the ER 7) Synthesised protein will then be transported via a vesicle to the Golgi complex for secretion or the lysosome 8) Signal sequence is cleaved and the SRP recycles once the polypeptide is completely synthesised within the ER

Question 41

what about membrane transporters e.g. Na+/K+ transporters

Answer 41

Proteins targeted for membrane fixation (integral proteins) get embedded into the ER membrane and ER membrane fuses with the celllmembrane over time