PW - Bacterial Porins Flashcards
What are the problems with membrane protein structure determination? (4)
X-ray crystallography
- Difficult to prepare crystals
Electron Microscopy (2D electron diffraction)
- Low to medium resolution
- Requires formation of 2D crystals
Indirect techniques (optical spectroscopy/mutagenesis expts)
NMR Spectroscopy
- Solution state NMR techniques – structure in micellar systems
- Solid state NMR techniques – structure in the bilayer
What are the 2 types of bacterial porins?
1) General diffusion (e.g. OmpF)
- Limited by Mw (e.g. OmpF),
- Rate proportional to concentration gradient
2) Substrate specific (e.g. LamB/Maltoporin)
- Recognize substrate
- Exhibit Michaelis-Menten kinetics
What are 4 features of OmpF
- 115 kDa, forms a homo-trimer
- 16 antiparallel strands, tilted by 45 degrees
- Loop 3 fold back into channel and has a highly conserved PEFGG motif
- Loop constricts pore to 15x22A.
What contributes to OmpF selectivity?
Mutation of D113 and E117 reduced conductivity by 50%
- Selectivity for cations is abolished
- MD show that these negative charges draw cations into the constriction site
What are 4 features of LamB/Maltoporin?
- 18 stranded porin
- Classical structure{short periplasmic loops and long extracellular loops
- Outer face of beta-barrel, covered with largely uncharged groups (grey – carbon)
- Pore constricted by 3 loops, confiring an hour glass shape to the pore
How does selectivity of LamB transport sugars?
Transport via a greasy slide
Six aromatic residues make up greasy slide
- Trp74 from adjacent monomer
- Tyr41, Tyr6, Trp420, Trp358, Phe227
Constricted by Tyr118 (green)
- Limits movement of sugars (selectivity)
Forms a smooth hydrophobic path which interactions with apolar surface of pyranose rings
- Sugar molecules slide along
How is the selectivity of sugars regulated? (4)
Ionic Tracks
- Three major sugar binding sites (S2-4)
- Aromatics interact with hydrophobic surface of sugars
- Hydrogen-bonding stabilizes the hydroxyl groups on the sugar
- Oligosaccharides can twist through the pore.
How are sugars transported? (2)
Via in-register shifts
- Sugars move along a network of H-bonds
- Specificity inferred through the position of H-bonds
What are exceptions related to VDAC- a mitochondrial beta barrel protein? (4)
- Endosymbiotic theory
Mitochondria – evolved from bacteria - Despite this VDAC encoded for by nuclear genome
- Mitochondrial OM does not contain LPS
- Very little variation between loops in VDACs
N-terminal extensions more common
What is the function of VDAC and how is its selectivity determined?
Involved in the transport of ATP and ADP across the outer membrane in mitochondria
- Pore lined with positive charge accounting for ATP/ADP (anion) selectivity
Beta barrels summary (5)
- General motif that is used in pro- and eukaryotes
- Highly stable architecture
- Folding of loops into the pore provides mechanisms for selectivity/regulation
- Can sustain high levels of transport
- Not particularly selective, allow leakage of ions
