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
