modelling membrane and protein structure (3)

Question 1

atomic resolution of membrane protein - number of membrane structures

Answer 1

number of membrane structures - increase exponentially

don’t readily form 3D and few structures - resolved by crystallography

Question 2

structures too large for liquid state NMR stages

Answer 2

find source of protein
isolate and purify
tools - enable to determine protein

Question 3

problem with membrane protein structure - protein expression

Answer 3

early structure solved

eukaryotic membrane

Question 4

protein expression -where early structure are solved

Answer 4

from natural abundant protein (mitochondria/chloroplast etc)

derived from bacteria

Question 5

protein expression - eukaryotic membrane

Answer 5

• expressed as heterologous in bacteria/yeast

- lacks post translation machinery for membrane expression and membrane insertion

Question 6

problem with membrane protein structure - solubility/purification/reconstitution

Answer 6

isolating protein - membrane proteins embedded - solubilising single chains and charged head groups using detergent molecules

Question 7

solubility/purification/reconstitution - stability after isolation protein

Answer 7

stability - very compromised - lose large quantity of protein as reconstitution remove detergent and add lipids - but lost asymmetry from original membrane so lots of info is lost

Question 8

problem with membrane protein structure - structural analysis methods

Answer 8

X-ray crystallography
NMR spectroscopy
Indirect technique
E- microscopy - 2D e- diffraction

Question 9

Structural analysis - X-ray crystallography

Answer 9

has protein and detergent micelles as they don’t crystallise as amount of protein exposed from crystal lattice - surface of protein - coated with detergent micelles

Question 10

Structural analysis - NMR spectroscopy

Answer 10

solution NMR - structure in micellar system

solid state NMR - structure in bilayer

Question 11

Structural analysis - indirect technique

Answer 11

optical microscopy/ mutagenesis/ model

Question 12

Structural analysis - E- microscopy (2D e- diffraction)

Answer 12

low to medium resolution

require formation of 2D crystal

Question 13

indirect technique in modelling

Answer 13

hydropathy plot
location of post-translational modification
labelling studies

Question 14

hydropathy plot

Answer 14

sequence analysis

reveal potential transmembrane helices but amphipathic helices - difficult to identify

Question 15

labelling studies

Answer 15

use membrane impermeant reagent - identify surface exposed external residue and HP labelling reagent
help determine transmembrane residue
can see whether residues are inside of outside of cell

Question 16

Hydropathy plot - process

Answer 16

each amino acid is assigned value corresponding in hydrophobicity
average length of transmembrane helix - 20-24 amino acids
identify HP sequence = high chance of forming -transmembrane if 20-24 long

Question 17

transmembrane organisation - sequence pattern

Answer 17

start with N-terminus - soluble amino acids
then 20-24 HP chain residue within bilayer
then more chain on other side with C end terminus

Question 18

glycosylation of membrane

Answer 18

able to identify motif - which terminus is on the outside
as process occurs outside
orientate integral protein

Question 19

example of glycosylation

Answer 19

glycophorin A - modified on extracellular surfaces
links always present on outside of bilayer
N-linked - glycosylation area - (Asn-X-Thr) or (Asn-X-Ser)

Question 20

glycosylation pattern

Answer 20

varies depending on pattern of inherited glycosylation enzyme - by an individual

Question 21

gives rise to what system in glycosylation

Answer 21

ABO blood group system

Question 22

X in N-linked glycosylation

Answer 22

another amino acid

Question 23

how hydropathy plot calculated

Answer 23

by computer algorithm

1-20, 2-21, 3-22 etc - revealing potential transmembrane helices

Question 24

model of rhodopsin

Answer 24

has 7 peaks in the hydropathy analysis - 7 transmembrane domain

Question 25

palmitoylation

Answer 25

addition of palmitoyl group - FA added to cysteine residue
anchor part of protein chain to surface of lipid bilayer
loop between peak 7 and C-terminus - for signalling

Question 26

structure of rhodopsin

Answer 26

first membrane protein solved as it was very abundant

contain retinal molecules - polyamine chain - absorb very strongly in visible range - easy to identify

Question 27

genomic analysis

Answer 27

can understand entire genome and identify frequency of no. domains
able to understand how different organisms start to work
good tool for identifying families of proteins

Question 28

applying hydropathy analysis - E coli

Answer 28

assimilate nutrient from surrounding - need transport
distribution of protein - higher on 12 transmembrane domain
evolved to reutilise 12 domain ‘scaffold’ to create transport protein
uses electrochemical gradient to assimilate sugar and amino acid surrounding

Question 29

applying hydropathy analysis - human

Answer 29

more in 7 transmembrane domain
for proteins like G-protein etc
not too many larger one due to cells specialising to breakdown nutrients

Question 30

beta(b)-barrel structure

Answer 30

form hydrophilic pores in outer membranes of bacteria and mitochondria

Question 31

how b-barrel formed

Answer 31

create energetically favourable protein structure - barrel structure with HP groups pointing out from surface

Question 32

HP groups of b-barrel pointing out

Answer 32

forms VdW between acyl group (HP) side chain and lipid

Question 33

maximising H-bonding potential in b-barrel

Answer 33

formed between NH and CO of backbone minimise charge - stable

Question 34

why hydrophilicity wont pick up b-barrel residue chains

Answer 34

b strand side chains - not always going to be on same face
HP amino acids can be in the centre
therefore barrel is coated with HP groups (greasy) interact with bilayer - anchor

Question 35

sucrose specific porin - function

Answer 35

allow sucrose to diffuse across outer membrane of bacterium a. typhimurium

Question 36

sucrose specific porin - structure

Answer 36

made up if 16 b strands - can vary and change size of porins

channel and in centre - charged groups are used to make hydrophilic channels

Question 37

b-barrel in hydropathy plot

Answer 37

no longer than 7 amino acid long

going up and down on hydrophobicity