membrane proteins 2

Question 1

most energetically favourable way to bury sequence in a hydrophobic environment is to

Answer 1

neutralise charged groups

Question 2

problems with membrane protein structure determination

Answer 2

expressed heterologously in bacteria and yeast

they have a lack of post transcriptional mod for efficient membrane protein expression

Question 3

problems with solubilization, purification, reconstruction

6points

Answer 3

affinity chromatography
-add detergent forming micelle spheres
-hopefully protein doesnt aggregate 
we have to remove the detergent to study it and add it to bilayer
no control over orientation 
50% protein back

Question 4

bacterial porins 1 classes what are they and

what are their facts

Answer 4

general diffusion (eg OMPF)

• limited by Mw
• rate proportional to conc. gradient
• limited by size and diffusion

Question 5

bacterial porins 1 classes what are they and

what are their facts

Answer 5

substrate specific (eg lamB / maltoporin)

• recognize substrate
• exhibits michaelis - menten kinetics
• saturated number of binding sites
• we can limit the amount of substrates that can go through

Question 6

OMPF a non specific porins
key facts
4 points

Answer 6

115Kda forms homo-trimer
16 antiparallel strands tilted by 45 degrees
loop 3 folds back into channel, has highly conserved PEFGG motif
loops constrict pore to 15 x 22AA

Question 7

LamB/ maltoporin substrate specific

facts

Answer 7

18 stranded porin
short periplasmic loop and long extracellular loop
outer face of B-barrel, has largely uncharged groups
pore restricted by loop 3, hour glass shape

Question 8

transport via greasy slide

what makes up the slide

Answer 8

6 aromatic residues
2 tyr, 3 trp, 1 phe
trp 118 constricts
forms hydrophobic pathway which interacts with apolar surface of pyranose ring

Question 9

ionic tracts
facts what are they
4 points

Answer 9

3 major sugar binding sites
aromatic res interact with hydrophobic residues of surface of sugar
h-bonding stabilizes the hydroxyl-group on the sugar
oligosaccharides can twist through the pore

Question 10

sugar transport comes via what

Answer 10

in registered shifts

Question 11

what are the in registered shifts

Answer 11

charged residues form specific binding sites

the oligosaccharides will bind and slide through for each reaction

Question 12

sugar moves along a network of H-bond this gives what

Answer 12

low energy barrier to transport

Question 13

lineweaver - burk plot what does it show

Answer 13

shows transport behaviour

Question 14

VDAC what is it

Answer 14

mitochondria B barrel protein

Question 15

VDAC what did it evolve from
where is it encoded
what does it not contain
variation between loops

Answer 15

mitochondria evolved from bacteria
nuclear genome
mitochondria OM doent contain LPS
very little variation in VDAC loops

Question 16

VDAC te channel itself facts

Answer 16

voltage gated channel -ve charged anions can go through it
n and c termini on opposite sides of the bilayer
n terminus relatively unsaturated - flexable

Question 17

selectivity

how is the pore selective

Answer 17

lined with +vely chnarged accounting for ATP/ADP -ve charge passing through it
2 different conformations, charge allows gating of the porin opening it

Question 18

potassium channels

Answer 18

highly efficient
1000 fold for K then Na
close to diffusion limit
opens by ph, voltage, ions, ligands g-proteins

Question 19

how have K+ channels appear to have been evolved

Answer 19

by addition of modular regulatory domains

Question 20

structural study of K+ what is done

Answer 20

expression
purification
crystallization
and structure

Question 21

structural study of K+

expression

Answer 21

channel expressed in homologous host

removed weakly ordered domains

Question 22

structural study of K+

purification

Answer 22

solubilized in decylmaltoside detergent
his tagged to aid purification
c-terminus removed, gel filtered, exchanged into LDAO

Question 23

structural study of K+

crystallization

Answer 23

many subsequent structures with Ab fragments to stabilize crystal

Question 24

structural study of K+

structure

Answer 24

x ray crystallography

Question 25

what is added to micelle to be found in crystals

Answer 25

antibiotics

mechanism to pull out this protein in 1 step

Question 26

ion conduction pathway

structure

Answer 26

4 binding sites for these K+ ions
5th is outside
no water around -which requires alot of energy
K+ ions are desolvated
hydrated K+ ion is located in a cavity in the centre of the bilayer

Question 27

ion selectivity

how is it done

Answer 27

replacing water ligand with protein ligand
backbone carbonyl groups which are interacting with K+
gap in Na large so larger energy barrier associated with it
2 K+ are bound at 1 time in between we get water molecules

Question 28

how does the K+ move through the ion channel

Answer 28

repulsion pushes the K+ out while another enters

because of conce gradient

Question 29

what is the vestibule

Answer 29

where K+ binds

Question 30

MthK is what and what regulate it

Answer 30

channel gating

regulated by Ca2+

Question 31

MthK regulatory domains

Answer 31

RCK Ca2+= sensing domain located on cytoplasmic face
4 copies per monomer, 2 attached to channel
makes gating ring of 8 RCK domains
alpha beta fold
cleft between 2 domains forms 2 Ca2+ binding sites

Question 32

Kv : S1-S4 WHAT DOES IT CONTAIN

Answer 32

voltage sensing domain

sense voltage cross bilayer movement from 1 side to another

Question 33

Kv : S1-S4

what is the sensor composed of

Answer 33

4 helical TM domains

sensitive to small mV changes

Question 34

structure of S1-S4s 4 TM domains

Answer 34

6 positively charged arg
pairing in the same direction
3 temp helices not alpha
can pair them with acid residues

Question 35

putative mechanism for voltage gating

Answer 35

apply voltage in bilayer
shuttling the charge from one residue to another
S4 helix and S5 links voltage gated domain with pore domain