modelling membrane and protein structure (3) Flashcards
atomic resolution of membrane protein - number of membrane structures
number of membrane structures - increase exponentially
don’t readily form 3D and few structures - resolved by crystallography
structures too large for liquid state NMR stages
find source of protein
isolate and purify
tools - enable to determine protein
problem with membrane protein structure - protein expression
early structure solved
eukaryotic membrane
protein expression -where early structure are solved
from natural abundant protein (mitochondria/chloroplast etc)
derived from bacteria
protein expression - eukaryotic membrane
- expressed as heterologous in bacteria/yeast
- lacks post translation machinery for membrane expression and membrane insertion
problem with membrane protein structure - solubility/purification/reconstitution
isolating protein - membrane proteins embedded - solubilising single chains and charged head groups using detergent molecules
solubility/purification/reconstitution - stability after isolation protein
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
problem with membrane protein structure - structural analysis methods
X-ray crystallography
NMR spectroscopy
Indirect technique
E- microscopy - 2D e- diffraction
Structural analysis - X-ray crystallography
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
Structural analysis - NMR spectroscopy
solution NMR - structure in micellar system
solid state NMR - structure in bilayer
Structural analysis - indirect technique
optical microscopy/ mutagenesis/ model
Structural analysis - E- microscopy (2D e- diffraction)
low to medium resolution
require formation of 2D crystal
indirect technique in modelling
hydropathy plot
location of post-translational modification
labelling studies
hydropathy plot
sequence analysis
reveal potential transmembrane helices but amphipathic helices - difficult to identify
labelling studies
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
Hydropathy plot - process
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
transmembrane organisation - sequence pattern
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
glycosylation of membrane
able to identify motif - which terminus is on the outside
as process occurs outside
orientate integral protein
example of glycosylation
glycophorin A - modified on extracellular surfaces
links always present on outside of bilayer
N-linked - glycosylation area - (Asn-X-Thr) or (Asn-X-Ser)
glycosylation pattern
varies depending on pattern of inherited glycosylation enzyme - by an individual
gives rise to what system in glycosylation
ABO blood group system
X in N-linked glycosylation
another amino acid
how hydropathy plot calculated
by computer algorithm
1-20, 2-21, 3-22 etc - revealing potential transmembrane helices
model of rhodopsin
has 7 peaks in the hydropathy analysis - 7 transmembrane domain
palmitoylation
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
structure of rhodopsin
first membrane protein solved as it was very abundant
contain retinal molecules - polyamine chain - absorb very strongly in visible range - easy to identify
genomic analysis
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
applying hydropathy analysis - E coli
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
applying hydropathy analysis - human
more in 7 transmembrane domain
for proteins like G-protein etc
not too many larger one due to cells specialising to breakdown nutrients
beta(b)-barrel structure
form hydrophilic pores in outer membranes of bacteria and mitochondria
how b-barrel formed
create energetically favourable protein structure - barrel structure with HP groups pointing out from surface
HP groups of b-barrel pointing out
forms VdW between acyl group (HP) side chain and lipid
maximising H-bonding potential in b-barrel
formed between NH and CO of backbone minimise charge - stable
why hydrophilicity wont pick up b-barrel residue chains
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
sucrose specific porin - function
allow sucrose to diffuse across outer membrane of bacterium a. typhimurium
sucrose specific porin - structure
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
b-barrel in hydropathy plot
no longer than 7 amino acid long
going up and down on hydrophobicity
