electron biomembranes - yeast cell tomographic...
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Biomembranes - Yeast Cell Electron tomographic
reconstructions
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Membrane proteins account for ~30%
of all proteins.
The fluid mosaic model of membrane structure proposed by S. J. Singer and G. L. Nicolson. Membrane is ~ 5nm thick.
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Membrane Proteins
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Sugars are covalently attached to certain amino acid side chains in regions that
face outside the cell.
a-helical transmembrane proteins
SH groups facing the cytoplasm are reduced (by glutathione in eucariotes and thioredoxin in
bacteria).
SH groups facing outside the cell are oxidized to S-S.
Polypeptide chain folds independently in these
three regions The major histocompatibility (MHC) antigen
HLA-A2
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POPC bilayer
The hydrogen bonds within the protein backbone and amino acids polarity are
the determining factor in the intramembrane protein segment.
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ΔGo→w = -1.36 log Kp
Each amino acid has different hydrophobicity
Hydrophobicity index is high for nonpolar side chains
Kp < 1 Hydrophobic compound
Kp > 1 Hydrophilic compound
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Hydrophobicity indices plotted against residue number gives a curve that is called hydropathy plot
But the reality is not so simple !!!
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The effect of lipid stress on transmembrane proteins
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External stretch force and/or amphipaths with positive or negative spontaneous curvature asymmetrically added to one leaflet can thin and bend the bilayer at the protein–lipid interface, changing the force vectors and therefore prefer a better matched protein conformation, say, the open
state.
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Mechanosensitive Channels as Osmotic Pressure Relief Valves
(Perozo and Rees)
Hierarchy of mechanically-gated channels. Gating tension of MscL serves to avoid membrane rupture.
MscL
MscK
MscS
(Sukharev et al.)
Key Question: How does mechanical tension couple to the conformational change ?
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The Kv1.2 expressed in an Sf-9 cell responds to voltage steps ranging from −100 to 40 mV. The responses in on-cell mode (A) are drastically different from those in the whole-cell mode (B). The normalized conductances are graphically compared in C.
The mechano-sensitivity of Kv
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Porins
Diameter 40 Å; 30 – 50 Å high; mass 30-50 kD.
Quaternary structure of bacterial porins
Membranes are spanned by β-strands of 9–11 residues with a tilt of 20–45o out of the TM axis.
The outer membrane of gram negative bacteria, or in the outer membranes of mitochondria in eukaryotes.
The hydrophobic, lipid-exposed surface of a β-barrel is about 27 Å thick.
= polar R group, = non-polar R group
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Membrane Proteins with β-Barrel Structure
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ABC transporters Largest class of membrane proteins known to pump various
substrates ranging from chloride ions to vitamin B12 into or outside of cell.
Vitamin B12 Transporter – BtuCD – a tetrameric protein
The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Locher KP, Lee AT, Rees DC, Science 2002 May 10;296(5570):1091-8
q Cystic fibrosis (CFTR) – cystic fibrosis transmembrane regulator -defect in ABC genes. q Cancer - multidrug resistance proteins (e.g. MDR1 and MDRP1-over expression in tumor cells causes resistance to chemotherapeutic agents). q Bacterial multidrug resistance - involved in export of harmful substances out of cell.
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Structure • Tetrameric protein • 2 membrane spanning domains (BtuC)
• 2 ATP binding cassettes (BtuD)
BtuC (membrane spanning domain)
BtuD(ABC Cassettes) located just below the membrane surface
BtuF periplasmic binding protein delivers B12 to the mouth of BtuC
This molecular assembly is ~90 Å tall, ~60 Å wide and 30Å thick.
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The transport mechanism for
Vitamin B12
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The heptamer is 100 Å tall and 100 Å at its widest point.
Non-constitutive membrane proteins
28 Å
α-HL can self-assemble into a lipid bilayer creating an aqueous nanopore (inside diameter ∼2 nm) across the lipid membrane.
Protein pore-forming toxin Alphahemolysin α-HL
Staphylococcus aureus
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αHL assembly The water-soluble form of the toxin (LukF)
Prepore The assembled heptamer
The membrane-bound monomer
αHL buries ≈154 residues in the membrane, the total effect on the decrease of free energy is so great that pore formation is essentially irreversible.