membrane proteins 2 Flashcards
most energetically favourable way to bury sequence in a hydrophobic environment is to
neutralise charged groups
problems with membrane protein structure determination
expressed heterologously in bacteria and yeast
they have a lack of post transcriptional mod for efficient membrane protein expression
problems with solubilization, purification, reconstruction
6points
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
bacterial porins 1 classes what are they and
what are their facts
general diffusion (eg OMPF)
- limited by Mw
- rate proportional to conc. gradient
- limited by size and diffusion
bacterial porins 1 classes what are they and
what are their facts
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
OMPF a non specific porins
key facts
4 points
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
LamB/ maltoporin substrate specific
facts
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
transport via greasy slide
what makes up the slide
6 aromatic residues
2 tyr, 3 trp, 1 phe
trp 118 constricts
forms hydrophobic pathway which interacts with apolar surface of pyranose ring
ionic tracts
facts what are they
4 points
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
sugar transport comes via what
in registered shifts
what are the in registered shifts
charged residues form specific binding sites
the oligosaccharides will bind and slide through for each reaction
sugar moves along a network of H-bond this gives what
low energy barrier to transport
lineweaver - burk plot what does it show
shows transport behaviour
VDAC what is it
mitochondria B barrel protein
VDAC what did it evolve from
where is it encoded
what does it not contain
variation between loops
mitochondria evolved from bacteria
nuclear genome
mitochondria OM doent contain LPS
very little variation in VDAC loops
VDAC te channel itself facts
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
selectivity
how is the pore selective
lined with +vely chnarged accounting for ATP/ADP -ve charge passing through it
2 different conformations, charge allows gating of the porin opening it
potassium channels
highly efficient
1000 fold for K then Na
close to diffusion limit
opens by ph, voltage, ions, ligands g-proteins
how have K+ channels appear to have been evolved
by addition of modular regulatory domains
structural study of K+ what is done
expression
purification
crystallization
and structure
structural study of K+
expression
channel expressed in homologous host
removed weakly ordered domains
structural study of K+
purification
solubilized in decylmaltoside detergent
his tagged to aid purification
c-terminus removed, gel filtered, exchanged into LDAO
structural study of K+
crystallization
many subsequent structures with Ab fragments to stabilize crystal
structural study of K+
structure
x ray crystallography
