Download Topology of membrane protein

Master Course Biomolecular Sciences: Part III:
Membrane biogenesis, protein folding and sorting
‘Topology of membrane proteins’
Antoinette Killian
Membrane proteins are abundant and important
About 1/4 of all genes encodes
for membrane proteins
• Smell
• Taste
• Vision
They are essential for all
sensory perceptions
• Hearing
• Touch
They function as receptors,
sensors, motors, pumps, channels,
enzymes…….
More than 50% of all new
drugs is targeted towards
membrane proteins
2
Examples of structures of membrane proteins
Bacteriorhodopsin
Chloride
channel
Ca-ATPase
What are the ‘rules’ for integration of proteins in
a membrane?
o What determines whether a newly synthesized
protein becomes a membrane protein?
General structural rules
Biosynthetic rules
o What determines its topology?
α-helix
Relevant features for transmembrane a-helix:
• Hydrophobicity
• Length
5
Transmembrane helices
Sequences of ~ 20 amino acids with sufficient
hydrophobicity
Some proteins form β-structures
β-strand
β-sheet
Can proteins span a membrane as β-strand or β-sheet? 7
Proteins can span the membrane as β-barrels
PORINS
8
Requirements for bèta-barrels??
Alternating hydrophilic/hydrophobic residues
Many strands that together can form a barrel
Length: 8-12 amino acids to span the bilayer
9
β-barrels only occur in specific membranes
(bacterial outer membrane, mitochondrial outer
membrane) where they form pores
Most proteins are present as α-helices
10
Can we predict whether a protein is a membrane
spanning protein?
11
Hydrophobicity scale:
‘Window’ of 20 amino acids
with sufficient hydrophobicity
can form transmembrane helix
12
Result bioinformatics
Preference of specific amino acids for
position within the membrane:
o hydrophobic part
o lipid/water interface
Many proteins have Trp at the lipid/water
interface
K+-CHANNEL OF STREPTOMYCES LIVIDANS
Doyle et al. (1998) Science 280 : 69 - 77
The lipid/water interface is a broad and special
region where many interactions can take place
Wimley and White, 2003
Wimley and White: interface scale
Stephen White: FEBS Letters
Fig. 1C, polarity at interface
AcWL-X-LL
Partioning of small peptides with guest residue in lipid bilayers
(model systems !)
Towards a new ‘membrane integration’ scale …
General rule:
A hydrophobic stretch of amino acids is required
of sufficient length and sufficient hydrophobicity
More accurate predictions can be made when
using appropriate calibration scales that also take
into account the positioning of specific residues
in the membrane
What are the ‘rules’ for integration of proteins in
a membrane?
o What determines whether a newly synthesized
protein becomes a membrane protein?
General structural rules
Biosynthetic rules
o What determines its topology?
How about ‘real life’?
Do the same rules apply for integration of newly
synthesized proteins in their native membrane?
Or: how does the translocon decide which sequences
should stay inside the membrane
How to test this?
Gunnar von Heijne:
- Prepare DNA construct of a
transmembrane protein to
which a hydrophobic segment
is attached
- Incorporate glycosylation
sites next to the hydrophobic
segment
-Express in an in vitro system
in the presence of microsomes
- Test membrane incorporation
by analyzing glycosylation
Conclusion: the same rules do apply for integration of
newly synthesized proteins in their native membrane as
for synthetic peptides in model membrane systems
(71K)
o What determines whether a newly synthesized
protein becomes a membrane protein?
o What determines its topology?
Proteins insert according to positive-inside rule:
positive charges stay in cytoplasm
outside
inside
++ ++
Bioinformatics studies:
++
Gunnar von Heijne
Proteins orient in membranes
according to the positive-inside rule
Why?????
Side chains that are positively charged
preferentially stay inside
Why not His, why not negatively charged chains?
Some considerations……………
Why positive inside rule?
1. pK values
Why positive inside rule?
1. pK values: basic residues difficult
to deprotonate
Why positive inside rule?
1. pK values: basic residues difficult
to deprotonate
2. Anionic lipids
Some lipids are anionic, others do not carry a net charge
Why positive inside rule?
1. pK values:
- basic residues difficult to deprotonate
2. Anionic lipids:
- binding of positively charged residues by
electrostatic interactions
Why positive inside rule?
1. pK values:
- basic residues difficult to deprotonate
2. Anionic lipids:
- binding of positively charged residues by
electrostatic interactions
3. ∆Ψ
A transmembrane potential exists across
some membranes
E.coli envelope
OM
PE 75%
PG 20%
CL 5%
+
IM
-
Why positive inside rule?
1. pK values:
- basic residues difficult to deprotonate
2. Anionic lipids:
- binding of positively charged residues by
electrostatic interactions
3. ∆Ψ:
- favorable electrostatic interactions
- electrophoretic effect
Why positive inside rule?
1. pK values:
- basic residues difficult to deprotonate
2. Anionic lipids:
- binding of positively charged residues by
electrostatic interactions
3. ∆Ψ:
- favorable electrostatic interactions
- electrophoretic effect
4. Potential within the membrane
Dipole effects in membranes
δ−
δ−
δ+
Orientation of
carbonyls induce
membrane dipole
Membranes are less permeable for cations compared to anions
Why positive inside rule?
1. pK values:
- basic residues difficult to deprotonate
2. Anionic lipids:
- binding of positively charged residues by
electrostatic interactions
3. ∆Ψ:
- favorable electrostatic interactions
- electrophoretic effect
4. Potential within the membrane
- positive inside, due to dipole effects
Proteins insert according to positive-inside rule:
positive charges stay in cytoplasm
outside
inside
++ ++
Bioinformatics studies:
++
Gunnar von Heijne
Proteins normally adopt one specific topology.
But perhaps not all proteins………
Structure of EmrE:
a multi-drug transporter
EmrE is a structural heterodimer:
both monomers have opposite topology
- EmrE: small multi-drug resistance (SMR) protein:
X-ray structure suggests two different
orientations, i.e. a structural heterodimer:
-This may be a ‘dual topology’ protein!!
- In some organisms, coexpression of two
homologous, but oppositely oriented proteins is
required for drug efflux
Dual topology somehow important for
functioning of SMR proteins
Identification and evolution of
dual-topology membrane proteins
Mikaela Rapp, Erik Granseth, Susanna Seppälä
and Gunnar von Heijne
Presentation: Melissa, Adam, and Rutger